Novel Method to Study Autonomic Nervous System Function and Effects

Home , Corpus spongiosum penis, Spinal cord

NOVEL METHOD TO STUDY AUTONOMIC NERVOUS SYSTEM FUNCTION

AND EFFECTS OF TRANSPLANTATION OF PRECURSOR CELLS ON

RECOVERY FOLLOWING SPINAL CORD CONTUSION INJURY

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the

Degree Doctor of Philosophy in the Graduate School of

The Ohio State University

Yvette Stephanie Nout, DVM MS

*****

The Ohio State University

2006

Dissertation Committee:

Professor Jacqueline C. Bresnahan, Adviser Approved by:

Professor Michael S. Beattie

Professor Lyn B. Jakeman ______Professor Stephen M. Reed Adviser Markus H. Schmidt, MD PhD Graduate Program in Neuroscience

ABSTRACT

Disruption of bladder and sexual function are major complications following spinal cord injury (SCI). To investigate these behaviors in a rat model of

SCI, we developed a method to monitor micturition and erectile events by telemetry. Pressure monitoring has been described for recording penile erections in awake rats and involves placement of a catheter into the corpus cavernosum of the penis. We developed a variation on this technique involving pressure monitoring within the bulb of the corpus spongiosum penis (CSP). Using this technique we can record both erectile and micturition events. This technique was validated in 10 male rats and we demonstrated that telemetric recording of CSP pressure provides a quantitative and qualitative assessment of both penile erections and micturitions. Subsequently we monitored CSP pressures in 7 male rats subjected to SCI. We demonstrated that monitoring of CSP pressure in conscious rats is a valuable and reliable method for assessing recovery of autonomic function. Although recovery of micturition occurs in rats following incomplete SCI, recovery is limited and voiding remains inefficient. Moreover, changes consistent with spasticity and/or hyperreflexia were determined through waveform analysis.

ii To repair the injured spinal cord cell transplant strategies are being

developed since they appear to reduce secondary damage and promote regeneration. We examined the effect of transplanting glial restricted precursor

(GRP) cells and elevating cyclic adenosine monophosphate (cAMP) concentrations on recovery of autonomic functions following SCI in 53 rats. When compared to an operated control group no beneficial effects of this cell transplantation technique were found with regards to behavioral outcomes.

Histopathological analysis showed survival, migration, and differentiation of GRP

cells within the spinal cord. Transplanted GRP cells were well integrated in the spared host tissue and animals that had received GRP cells had more tissue throughout the lesion region and at the lesion center. Although elevation of cAMP appeared to reduce the area of spinal cord occupied by graft tissue, cAMP favored differentiation of GRP cells into oligodendrocytes. The transplantation technique used here did not affect serotonergic input to the neurons in the lumbosacral spinal cord that regulate micturition and sexual behavior.

iii

This thesis is dedicated to my parents

Robert and Frances Nout who taught me the value of education and perseverance.

“Carpe Diem”

ACKNOWLEDGMENTS

None other than Stephen Reed can top the list of people I would like to

take the opportunity here to thank. Thank you for teaching me the details of

equine neurology and for encouraging me to work with Drs. Beattie and

Bresnahan. Thank you for your continuous support and advice throughout the

past 6 years. I have enjoyed working with you tremendously and I hope we will

continue to do so for years to come. I would like to thank Michael Beattie and

Jacqueline Bresnahan, my advisors, for taking a chance on a veterinarian and allowing me to be part of their laboratory. It has been an extremely educational

and rewarding experience in an area I consider to be very meaningful to

veterinary and human medical progress. Thank you for this inspiring opportunity.

I would like to thank Markus Schmidt for teaching me so much about autonomic

behaviors and methods of examining these. I have enjoyed the enthusiasm and

energy that you have brought towards all aspects of these experiments. Thank

you for image preparation, your expertise, and support.

The work presented in this thesis could not have been completed without

the input of Amy Tovar, Rochelle Deibert, Crystal Forrider, Esther Culp, John

Komon, and the rats. Amy, I want to thank you very much for the many most

v excellent ideas you have put forward towards conducting these experiments and for teaching me, an equine internist, the techniques of rat microscopic surgery. I also want to thank you for your support inside the lab and at J.R. Miggs. I would like to thank Rochelle and Crystal for their help with behavioral testing, animal care, and tissue processing. Ladies, your work has been truly fabulous! Esther, I would like to thank you for all the hours of data analysis you have performed for these experiments. I have appreciated your hard work and I would like to thank you for always being available for questions and updates. I would like to thank

John Komon for help with histological analyses and excellent image preparation.

I would like to thank Margot Mayer-Proschel, Christoph Proschel, and Mark

Noble from University of Rochester Medical Center for providing me with the

GRP cells. I thank the rats for their participation in these experiments; rats are indeed very friendly creatures and play such an important role in advancement of medical research. They deserve appropriate protection through institutional animal care and use regulations such as present at The Ohio State University.

I would like to thank Lyn Jakeman for her enthusiasm, interest, and input in my work. You have been there through first year NGSP presentations, my candidacy examination, and finally this thesis. I would like to thank Richard Burry and William Muir for their insights in aspects of histology and cardiovascular regulation, respectively. I would like to thank both of you for your support during my candidacy examination.

I would also like to thank Dana McTigue and Ping Wei for their help with immunohistochemical staining techniques and Brian Kemmenoe and Kathy

vi Wolken for their expertise and help while teaching me to use the confocal microscope in the Campus Microscopy and Imaging Facility.

Now I would like to thank John Gensel, Brandon Miller, Fang Sun, Adam

Ferguson, and Randolph Christensen for their help and support in the lab during the past years. John, Brandon, and Adam, I have always enjoyed your company and all our conversations inside and outside the lab. Guys, keep up the good work and Adam, rock on! I would also like to thank Georgeta Mihai, Megan

Detloff, and Richa Tripathi for their support and friendship during the past years.

It has been great meeting all of you and communicating and working with you.

I would like to acknowledge advice, support, and friendship from Kenneth

Hinchcliff, Ramiro Toribio, Colin Schwarzwald, and Catherine Kohn. Ken, thank you for introducing me to basic research, for teaching me to write, and for continued much appreciated advice! Ramiro and Colin, thank you for all your encouragement and friendship! Of course I have loved our visits to Rose and

Thistle and Brazenhead and I hope we will continue to frequent these places. I would also like to thank my friends Cheryl Sofaly, Christopher O’Sullivan, Sue

Hunter, Laura Werner, Wyatt Winchell, Jamie Murphy, Cheney Meadows, Phillip

Lerche, Andrew Phipps, Christopher Premanandan, Wendy Fife, Mark Zuck,

Allison Stewart, Monica Figueiredo, Emma Rowe, Kevin Corley, Kristy Amer, and members from the Columbus Running Club. I appreciate all the support and I look forward to meeting up with all of you who have left me behind in Columbus! I would also like to take this chance to thank my friends in The Netherlands, Eva

Wolking, Stephen Postma, Angela and Arjen Ruiter, Inge Luning, Saxa van

vii Eijnsbergen, Annet van Uchelen, Corine Visser, Edwin Enzerink, and my Dutch

friends abroad, Nienke Zwank-Wijnia, Martine de Wit, and Ingrid Segboer. Jullie hebben allemaal bijgehouden waarmee ik me heb bezig gehouden de afgelopen paar jaar, en ik wil jullie bedanken voor jullie steun en alle gezellige reunies die we hebben kunnen hebben tijdens mijn bliksem bezoeken aan Wageningen.

Finally I want to thank my family in The Netherlands and Canada, my

brother Remi Nout, Maartje Schaap, and my parents for their unfailing love and

support. Remi en Maartje, bedankt voor alle gezelligheid in Amsterdam en Remi,

wees niet bang, jij blijft natuurlijk de enige echte Nout – dokter. Lieve ouders,

vanaf het moment dat ik dierenarts wilde worden hebben jullie mij niet alleen

altijd volledig gesteund, maar hebben jullie je ook actief ingezet en mij veel

mogelijkheden geboden zodat ik nu mijn dromen heb kunnen realiseren. Wie

had ooit gedacht dat ik mijn vaders voorbeeld zou volgen in het onderzoek??

Jullie hebben me zien vertrekken naar de UK, Duitsland, Tanzania, Virginia en

nu Ohio en gelukkig zijn jullie me vaak in de VS komen opzoeken. Dat er nog

veel vakanties zullen volgen! Ik wil jullie bij deze ontzettend bedanken voor jullie

liefde, steun, vakantie lol, en voor alles waar ik nu niet eens aan denk.

Thank you!

Dank jullie wel!

viii VITA

September 16, 1973 ...... Born – Bamako, Mali

1996 ...... MS, Veterinary Science, Utrecht University, The Netherlands

1999 ...... DVM, Utrecht University, The Netherlands

1999 – 2000 ...... Intern Equine Medicine and Surgery, Marion duPont Scott Equine Medical Center, Leesburg, Virginia, USA

2000 – 2003 ...... Resident Equine Medicine and Graduate Student, The Ohio State University, Ohio, USA

2003 ...... MS, Veterinary Clinical Sciences, The Ohio State University, Ohio, USA

2004 ...... Diplomate, American College of Veterinary Internal Medicine

2003 – present ...... Graduate Research Associate, The Ohio State University, Ohio, USA

PUBLICATIONS

1. Newcombe J.R., Nout Y.S. (1998) Apparent effect of management on the hour of parturition in mares. Veterinary Record 142: 221 – 222.

2. Murray, M.J.; Nout, Y.S.; Ward, D.L. (2001) Endoscopic findings of the gastric antrum and pylorus in horses: 162 cases (1996-2000). Journal Veterinary Internal Medicine 15:401 – 406.

ix 3. Nout, Y.S.; Hinchcliff, K.W.; Samii, V.F.; Kohn, C.W.; Jose-Cunilleras, E. and Reed, S.M. (2002) Chronic pulmonary disease with radiographic interstitial opacity (interstitial pneumonia) in foals. Equine Veterinary Journal 34 (6) 542 – 548.

4. Nout, Y.S.; Corley, K.T.T.; Donaldson, L.L.; Furr, M.O. (2002) Indirect oscillometric and direct blood pressure measurements in anesthetized and conscious neonatal foals. Journal of Veterinary Emergency and Critical Care 12 (2) 75 – 80.

5. Nout, Y.S. and Reed, S.M. (2003) Cervical vertebral stenotic myelopathy. Equine Veterinary Education 5 (4) 268 – 281.

6. Nout, Y.S.; Hinchcliff, K.W.; Bonagura, J.D; Meurs, K.M.; Papenfuss, T.L. (2003) Cardiac amyloidosis in a horse. Journal Veterinary Internal Medicine 17 (4) 588 – 592.

7. Nout, Y.S.; Hinchcliff, K.W.; Jose-Cunilleras, E.; Dearth, L.R.; Sivko, G.S.; DeWille, J.W. (2003) Moderate exercise and hyperglycemia after exercise do not alter muscle GLUT-4 expression in horses. American Journal of Veterinary Research 64 (11) 1329 – 1456.

8. Jose-Cunilleras; Hinchcliff, K.W.; Nout, Y.S.; Geor, R.J. (2004) Glucose kinetics in septic neonatal foals. Journal Veterinary Internal Medicine 18 (3) 412.

9. Matthews, H.K.; Nout, Y.S. Spinal cord, vertebral, and intracranial trauma. In: Reed, S.M.; Bayly, W.M.; Sellon, D.C. eds. Equine Internal Medicine 2nd ed. St. Louis, Missouri: Elsevier (USA), 2004.

10. Matthews, H.K.; Nout, Y.S. Equine degenerative myeloencephalopathy. In: Reed, S.M.; Bayly, W.M.; Sellon, D.C. eds. Equine Internal Medicine 2nd ed. St. Louis, Missouri: Elsevier (USA), 2004.

11. Nout, Y.S. Equine Motor Neuron Disease. In: Reed, S.M.; Bayly, W.M.; Sellon, D.C. eds. Equine Internal Medicine 2nd ed. St. Louis, Missouri: Elsevier (USA), 2004.

12. Nout, Y.S. Equine Grass Sickness. In: Reed, S.M.; Bayly, W.M.; Sellon, D.C. eds. Equine Internal Medicine 2nd ed. St. Louis, Missouri: Elsevier (USA), 2004.

13. Nout, Y.S.; Beattie, M.S.; Bresnahan, J.C.; Tovar, C.A.; Culp, E.; Schmidt, M.H. (2004) Telemetric monitoring in conscious rats of micturition and

x penile erectile events using corpus spongiosum penis pressures following spinal cord injury. J Neurotrauma 21:1271.

14. Nout, Y.S.; Beattie, M.S.; Bresnahan, J.C.; Tovar, C.A.; Culp, E.; Schmidt, M.H. (2004) Telemetric monitoring in conscious rats of micturition and penile erectile events using corpus spongiosum penis pressures following spinal cord injury. Program No. 922.17. 2004 Abstract Viewer/Itinerary Planner. Washington, DC, Society for Neuroscience, Online.

15. Schmidt, M.H., Nout, Y.S., Culp, E., Tovar, C.A., Beattie, M.S., Bresnahan, J.C. (2004) A new technique for the simultaneous recording of micturition and penile erections in freely behaving rats: Telemetric monitoring of corpus spongiosum penis (CSP) pressure. Program No. 922.16. 2004 Abstract Viewer/Itinerary Planner. Washington, DC, Society for Neuroscience, Online.

16. Nout, Y.S.; Schmidt, M.H; Tovar, C.A.; Culp, E.; Beattie, M.S.; Bresnahan, J.C. (2005) Telemetric monitoring of corpus spongiosum penis pressure in conscious rats for assessment of micturition and sexual function following spinal cord injury. J Neurotrauma 22 (4) 429 – 441.

17. Nout, Y.S.; Bucellato, M.; Oglesbee, M.; Reed, S.M. (2005) Polyneuritis equi: 7 cases on 1 farm. Journal of Veterinary Emergency and Critical Care 15 (3) Supplement 1, S11.

18. Nout, Y.S.; Leedy, G.M.; Beattie, M.S.; Bresnahan, J.C. Alterations in eliminative and sexual reflexes after spinal cord injury: defecatory function and development of spasticity in pelvic floor musculature. In: Weaver, L. eds. Progress in Brain Research 152, 359 – 372. Amsterdam, The Netherlands: Elsevier, 2005.

19. Nout, Y.S. and Reed, S.M. (2005) Examination and treatment of the recumbent horse. Equine Veterinary Education 7 (6) 416 – 432.

FIELDS OF STUDY

Major Field: Neuroscience

xi TABLE OF CONTENTS

ABSTRACT…………………………………………………………………………ii

DEDICATION……………………………………………………………………….iv

ACKNOWLEDGMENTS……………………………………………..…………….v

VITA………………………………………………………………………………….ix

LIST OF TABLES………………………………………………………………….xiv

LIST OF FIGURES…………………………………………………………………xv

CHAPTERS

1. Introduction……….………………………………………………………..1

2. Alterations in eliminative and sexual reflexes following spinal cord injury…………………………………………………………. 9 2.1 General characteristics……………………………………………..9 2.2 Micturition……………………………………………………………24 2.3 Defecatory function………………………………………………...28 2.4 Sexual function……………………………………………………..31 2.5 References…………..……………………………………………..35

3. A novel technique for monitoring micturition and sexual function in male rats using telemetry…………………………………42 3.1 Introduction………………………………………………………….42 3.2 Materials and methods…………………………………………….48 3.3 Results………………………………………………………………56 3.4 Discussion…………………………………………………………..67 3.5 References………………………………………………………….72

xii 4. Telemetric monitoring of corpus spongiosum penis pressure in conscious rats for assessment of micturition and sexual function following spinal cord contusion injury..……………………75 4.1 Introduction…………………………………………………………..75 4.2 Materials and methods……………………………………………..78 4.3 Results……………………………………………………………….83 4.4 Discussion…………………………………………………………...94 4.5 References…………………………………………………………101

5. Effects of transplanting glial restricted precursor cells and elevating cAMP concentrations on autonomically regulated behavior and histopathological outcomes in male rats following spinal cord contusion injury…………………104 5.1 Introduction…………………………………………………………..104 5.2 Materials and methods……………………………………………..109 5.3 Results……………………………………………………………….126 5.4 Discussion…………………………………………………………...154 5.5 References…………………………………………………………..166

6. Discussion…………………………………………………………………..173

APPENDIX……………………………………………………………………………179

Images from spinal cord sections……………………………………....179

REFERENCES……………………………………………………………………….185

xiii LIST OF TABLES

Table Page

3.1 Micturition waveform characteristics in the adult Long-Evans rat...... 59

3.2 Erectile event waveform characteristics in the adult Long-Evans rat...... 66

4.1 Micturition waveform characteristics...... 89

4.2 Characteristics of full erectile event waveforms...... 91

5.1 Outline of experimental groups...... 110

5.2 List of antibodies used for immunohistochemistry...... 122

xiv LIST OF FIGURES

Figure Page

2.1 The caudal lumbar and rostral sacral spinal cord contains the neuronal circuitry that organizes the parasympathetic components of bladder, bowel and sexual function...... 13

3.1 Electromyographic activity of the BS and IC muscles during erectile events...... 44

3.2 Telemetry system diagram...... 47

3.3 TA11PA-C40 pressure transducer used in these studies...... 49

3.4 Rat penile anatomy...... 50

3.5 Transducer implantation procedure...... 51

3.6 Experimental condition for telemetry recording...... 52

3.7 Ex copulatory reflex erection test...... 54

3.8 CSP pressure waveform obtained by telemetry during micturition...... 58

3.9 Electromyographic activity in the BS muscle during a micturition event...... 59

3.10 Number of micturitions...... 60

3.11 Total number of micturitions seen on video and detected by telemetry...... 60

3.12 Bland and Altman plot of the number of micturitions per 24 hours detected by video or telemetry...... 61

xv 3.13 Relationship between duration and volume of micturition...... 62

3.14 CSP pressure waveform obtained by telemetry during a full erectile event...... 64

3.15 CSP pressure waveform obtained by telemetry during a partial erectile event...... 64

3.16 CSP pressure waveforms recorded during 3 behavioral contexts...... 65

4.1 Representative waveforms of erectile and micturition events. . . .85

4.2 Representative micturition waveforms following SCI...... 86

4.3 Total number of micturitions following SCI...... 87

4.4 Urine volume per micturition...... 88

4.5 Total number of erectile events following SCI...... 90

4.6 Reflex erection test results...... 92

4.7 Recovery of locomotion function...... 93

4.8 Spared tissue at lesion epicenter...... 94

5.1 Time line for surgical manipulations and administration of treatments...... 111

5.2 Site of injections made into the dorsal spinal cord during the transplant procedure...... 115

5.3 Impact parameters...... 127

5.4 Recovery of locomotion function...... 129

5.5 Activity of all rats in their cages...... 129

5.6 Recovery of micturition function shown for all groups...... 131

5.7 Duration of micturition events...... 133

5.8 Frequency of micturition pressure peaks...... 134

xvi 5.9 Mean pressure during micturition events...... 135

5.10 Number of full erectile events...... 137

5.11 Duration of full erectile events...... 138

5.12 Maximum pressure during full erectile events...... 139

5.13 Number of partial erectile events...... 141

5.14 Latency during the reflex erection test...... 142

5.15 Number of peaks per erectile event...... 143

5.16 Thoracic spinal cord stained for GRP cells 48 hours after cell transplant...... 144

5.17 Illustration of lesion center and lesion region...... 145

5.18 Representative lesion centers for all groups...... 146

5.19 Tissue at lesion center...... 147

5.20 Tissue in lesion region...... 149

5.21 Area of hPLAP positive cells in the lesion region...... 149

5.22 Differentiation of hPLAP positive GRP cells...... 151

5.23 Quantification of differentiation of hPLAP positive GRP cells. . . 152

5.24 Dorsomedial and dorsolateral nuclei in the lumbosacral spinal cord...... 153

5.25 Serotonergic input to motor neurons in the lumbosacral spinal cord...... 153

5.26 Motor neuron cells found in the dorsomedial and dorsolateral nuclei...... 154

A.1 Representative images of animals that received GRP cell transplants – transverse sections lesion region...... 179

A.2 Representative images of animals that received GRP cell transplants – horizontal sections lesion region...... 182

xvii CHAPTER 1

INTRODUCTION

Acute traumatic damage to the spinal cord occurs in most species, but is most commonly seen in humans, dogs, and horses. In dogs the most common causes of spinal cord injury (SCI) are intervertebral disk extrusion and traumatic penetrating or non-penetrating injuries (LeCouteur and Grandy, 2000). Explosive protrusion of an intervertebral disk can cause both concussion and compression of the spinal cord. Disk extrusion, a condition that occurs in many breeds of dogs, may occur at any site within the vertebral column but is most commonly seen in the thoracolumbar and cervical area of the vertebral column. It has been reported that 1 – 2% of all dogs admitted to veterinary hospitals have injuries to the spinal cord resulting from disc disease (Hoerlein 1953; Priester 1976, Webb et al.,

2004) but total incidence of SCI in hospitalized dogs has not been reported. In horses, trauma to the central nervous system is the most common cause of neurologic disease, accounting for 22% of neurologic disorders in one study

(Feige et al., 2000). In this same study, 50% of horses with traumatic central nervous system disease were diagnosed with lesions in the cervical spinal cord.

Spinal cord injury occurs predominantly in young horses and is most

commonly the result of falls (Matthews and Nout, 2004; Reed, 1994). In horses the spinal cord is well protected in the thoracic and lumbar vertebral column, leaving the cervical and sacral vertebral column the most susceptible to trauma.

There are no reports on incidence of SCI in horses; however, since in general

SCI is the result of high velocity / high impact forces, prognosis for life is guarded. In humans, it is estimated that the annual incidence of SCI, not including those who die at the scene of the accident, is approximately 40 cases per million population in the U.S., or approximately 11,000 new cases each year.

The number of SCI patients in the U.S. who were alive in July 2005 was estimated to be approximately 250,000. Between 2000 and July 2005 vehicular crashes accounted for the majority (47.5%) of reported SCI cases, followed by falls (22.9%) and violence (13.8%) (The National SCI Statistical Center, 2005).

Spinal cord injury results not only in paraplegia or tetraplegia, but also leads to systemic and metabolic alterations secondary to disruption of autonomic nervous system homeostasis. Currently, in a time of increasing emotional value of many of our pets, treatment and care of SCI and other recumbent patients has become an important aspect of veterinary medicine (Nout and Reed, 2005).

Although recovery from incomplete SCI in small animals is good, with the majority of dogs regaining motor function, animals with more severe injuries have a much poorer prognosis (Kinzel et al., 2005; LeCouteur and Grandy, 2000;

Olby, 1999; Rossmeisl et al., 2005). Furthermore, mobility of paraplegic dogs is improved through development of pet wheelchairs and dog carts; however, it is dysfunction of eliminative behaviors, or incontinence, which poses more serious

problems in the care of these animals. Despite advances in the development of

assistive devices these additional consequences of SCI pose threats that

ultimately may lead to the decision that favors humane euthanasia. In one study

examining long-term (> 6 months) functional outcome after severe SCI in 87

dogs, the prognosis for paraplegic dogs without deep pain perception because of

trauma was guarded, while dogs with disk herniation had a better chance of

recovering motor function (Olby et al., 2003). A third of the dogs that recovered

motor function had intermittent incontinence. Persistent loss of deep pain

perception did not preclude recovery of motor function, but such dogs remained

incontinent.

In humans, since 2000, the average age at injury was 38 years and 79.6%

of spinal cord injuries occurred in males. Thus, primarily young male adults are

affected. Since 2000, the most frequent neurologic category at the time of

discharge from hospital was incomplete tetraplegia (34.5%), followed by

complete paraplegia (23.1%), complete tetraplegia (18.4%), and incomplete

paraplegia (17.5%) (The National SCI Statistical Center, 2005). This indicates

that 76 – 93.5% of SCI patients are left with incomplete function below the waist which includes inability to walk, loss of bladder and defecatory control, and altered sexual function. In fact, regaining bladder/bowel function was the first or

second highest priority for 39.7% of quadriplegics and 38% of paraplegic.

Similarly, regaining sexual function was the first or second highest priority to

28.3% of quadriplegics and 45.5% of paraplegics (Anderson, 2004).

These changes profoundly reduce the quality of life for injured individuals

and negatively impact society by reducing productivity in injured individuals. The

average yearly health care, living expenses, and the estimated lifetime costs that

are directly attributable to SCI vary greatly according to severity of injury but exceed $7 billion dollars per year in the U.S. The additional indirect costs including losses in wages, fringe benefits, and productivity average $56,964 per person per year (Center, 2005; DeVivo, 1997).

Damage inflicted to the spinal cord occurs in 2 distinct phases. So-called

“primary injury” refers specifically to the mechanical disruption of axons as a

result of compression, penetration, laceration, shear, and/or distraction (Tator

and Fehlings, 1991). Primary damage results in cell membrane disruption and

rapid cell death which is followed by a second period of progression of tissue

damage and cell death that can last several hours to days after initial insult

(Allen, 1911, 1914; Beattie et al., 2000a; Beattie et al., 2000b; Tator and

Fehlings, 1991). This is referred to as “secondary injury”. Many well-

characterized mechanisms of cell damage have been shown to play a role during

this period of secondary damage, including 1) vascular compromise leading to

reduced blood flow, loss of autoregulation, loss of microcirculation, vasospasm,

thrombosis, and hemorrhage; 2) electrolyte shifts, permeablility changes, loss of

cellular membrane integrity, edema, and loss of energy metabolism; 3)

biochemical changes including neurotransmitter accumulation, arachidonic acid

release, free radical and prostaglandin production, and lipid peroxidation; and 4)

apoptosis (Amar and Levy, 1999; Beattie et al., 2000a; Hall and Wolf, 1986;

Tator, 1996, 1998; Tator and Fehlings, 1991). Secondary expansion of the lesion

cavity that occurs through necrotic and apoptotic pathways is associated with the

invasion of immune cells and activation of the resident microglial population. The

period of ongoing secondary injury is considered to be the phase in which

therapeutic measurements could be taken to halt the process of ongoing cell loss.

Endogenous repair has been shown to take place following a contusion

lesion. Proliferation of ependymal cells that lie around the central canal has been

shown to occur in fetal mammals, but also in adult rats (Beattie, et al., 1997;

Mothe and Tator, 2005; Namiki and Tator, 1999; Wallace et al., 1987; Vaquero et

al., 1981). These cells appear to add tissue bridges referred to as trabeculae that

begin to fill the lesion cavity (Bresnahan, 1978; Bresnahan et al., 1987;

Bresnahan et al., 1976; Noble and Wrathall, 1989). Glial cells and invading

sensory axons can follow these cellular tracks. Over time, these trabeculae can

support growth of axons from within the central nervous system, such as the

corticospinal tract and fibers from the brainstem reticular formation (Beattie, et

al., 1997; Hill et al., 2001). In distinct contrast to the limited distribution of

neuronal stem cells, glial precursors are widely distributed in the developing and

mature mammalian central nervous system. Furthermore glial progenitor cells

that can give rise to both astrocytes and oligodendrocytes exist predominantly in

the outer rim of the spinal cord (Horner, et al., 2000). Proliferation of endogenous

glia and/or glial precursor cells is thought to play an important role in restoring

chronic function to the injured cord (Beattie et al., 1997; Beattie et al., 2002;

McTigue et al., 2001; Zai and Wrathall, 2005). It is likely that oligodendrocyte precursor cells rather than surviving mature oligodendrocytes contribute to the intrinsic remyelination after demyelinating insult (Cao et al., 2002).

Oligodendrocytes, the cells that wrap axons in myelin and allow them to function, undergo apoptosis during the delayed phase of death after SCI and since each oligodendrocyte myelinates 10 – 20 different axons, the resulting loss of myelination and conductance through that segment has a large effect on the loss of function (McDonald et al., 2004). Although remyelination occurs, this intrinsic repair is insufficient to overcome the insult. However, perhaps by increasing the number of these cells in the vicinity of the lesion, functionally significant repair could be obtained. Currently the use of various cell transplant procedures is being widely investigated in the field of SCI research. Rationales for using transplantation strategies are 1) the functional reconstruction of neuronal circuits;

2) the production of neurochemically active substances; and 3) remyelination of axons (Bjorklund and Lindvall, 2000; Okano, 2002).

Although it is currently possible to make axons sprout from surviving but damaged neurons, it is not yet possible to make long axons grow and form appropriate reconnections, however, this ultimate goal may be achievable through ongoing studies and investigations into the potential of stem cell transplantation in neurologic diseases. At this time, it appears that optimizing the function of neuronal circuits that survive SCI is probably the most achievable strategy (McDonald et al., 2004). Many current investigations attempt to do this through enhancement of glial cell function. Immature astrocytes promote axonal

growth and perhaps promote survival after injury (Silver, 1993), whereas

immature oligodendrocytes provide remyelination after injury (Kohama et al.,

2001; McTigue et al., 2001).

It has now become possible to isolate pluripotent cells from embryos and using treatments with growth factors, push these cells into lineage-specific states that produce either neuronal or glial-specific progenitor cells (Mayer-Proschel et al., 1997; Wu et al., 2002). For cell replacement in neurological conditions, experimental data to date suggests that lineage-committed immature neuronal or glial progenitor cells, rather than multipotential neural stem cells or mature

neurons and glial cells, are most likely to result in predictable phenotypic

differentiation and potential functional outcome. Key in these treatment strategies

is the ability of cells to integrate into the neural circuitry in a functional manner

and consequently contribute to the correction of functional deficits.

To improve integration and function of transplanted cells a wide variety of

bio-molecular therapies are being investigated. For example neurotrophic factors,

such as neurotrophins, have been found to promote a variety of neural

responses such as survival and outgrowth of nerves and spinal cord nerve

regeneration (Schmidt, C. E. and Leach, 2003). Inducing up-regulation of

neurotrophins, regeneration-associated genes, and antiapoptotic factors and / or

blocking inhibitory biomolecules in the nervous system through use of genetically

modified cells are methods used to complement cell transplantation strategies.

Elevation of cyclic adenosine monophosphate (cAMP) concentrations has been shown to play an important role in regeneration of the adult central nervous

system (Spencer and Filbin, 2004) and in overcoming inhibitory aspects of the injured spinal cord milieu (Gao et al., 2004). Recently, elevation of cAMP through administration of the phosphodiesterase IV inhibitor rolipram in conjunction with transplantation of embryonic spinal tissue or Schwann cells resulted in improved functional outcome and promoted axonal regeneration after SCI (Nikulina et al.,

2004; Pearse et al., 2004).

Our studies were undertaken to contribute to improving quality of life for all involved in SCI. The main focus of our research was dysfunction of eliminative and sexual function and effects of a cutting-edge transplantation strategy on these body systems. In the following investigations we demonstrate the value of a novel method to assess autonomic behavior in freely moving rats and use that model to investigate the use of a cell transplantation technique in experimental

SCI. Since the spinal cord injured population considers eliminative and sexual function to be very important in quality of life and these are important functional outcomes in veterinary spinal cord injured patients, we chose bladder and sexual functions to be part of the behavioral outcomes in our rat model of SCI. Using a telemetric technique to measure corpus spongiosum penis pressure we demonstrate the ability to assess details of micturition and erectile function in conscious male rats. We then use this technique to assess recovery of these functions after an intervention that includes a combination of transplantation of glial restricted precursor cells and elevation of cAMP concentrations.

CHAPTER 2

ALTERATIONS IN ELIMINATIVE AND SEXUAL REFLEXES AFTER SPINAL

CORD INJURY

2.1 General characteristics

Both eliminative and sexual functions are compromised following spinal

cord injury (SCI) (Comarr, 1970; Frenckner, 1975; Pedersen, 1983). Clinical

representations with regards to bladder and bowel function are detrusor

hyperreflexia, detrusor sphincter dyssynergia, detrusor areflexia, gastric

dilatation, ileus, fecal incontinence and constipation. Inability to achieve

psychogenic and reflexogenic erections and ejaculatory dysfunction in men and failure of vaginal lubrication in women are aspects that have a significant impact

on sexual life after SCI (Karlsson, 2005). Disruption of supraspinal input to the

neuronal circuits that control these functions is the main cause of the autonomic sequelae of SCI.

The eliminative and sexual function systems have both autonomic and

somatic components, and are organized by sympathetic neuronal pools in the

caudal thoracic/upper lumbar cord, parasympathetic neuronal pools in the sacral cord and somatic motor pools in the caudal lumbar/upper sacral cord. The

anatomical organization of these systems is co-extensive, their functions overlap,

and response to injury is similar. Although the basic afferent and efferent limbs of

the reflex components are segmentally organized, the loss of brainstem and

forebrain descending input results in loss of voluntary control and in abnormal

functioning of these systems including the development of dyssynergias and

spasticity (Nout, Y. S. et al., 2005a). Before reviewing aspects specific to the

different eliminative and sexual functions, in the following paragraphs the

characteristics that are similar for these functions are discussed. The most

important components of the innervation of rectum, lower urinary tract, and

genital tract are through the pelvic, hypogastric, and pudendal nerves.

Neuroanatomy

The pelvic plexus is a crossroad of autonomic nerves and ganglia

supplying the rectum, the lower urinary tract, and the genital tract (Paxinos,

1995). In the male rat, the main components of this plexus are the right and left

pelvic ganglia, also referred to as the hypogastric ganglia, or the major pelvic

ganglia. These ganglia are positioned on either side of the prostate, ventral to the

rectum, and caudal to the ureter and vas deferens, and are unusual among

autonomic ganglia, since they contain both parasympathetic and sympathetic

postganglionic neurons. The main afferents to the pelvic ganglia are the hypogastric nerve and the pelvic nerve (Paxinos, 1995).

The majority of fibers comprising the hypogastric nerve originate in the

intermediolateral cell column of the thoracolumbar cord, (thoracic (T)11 – L2

(Baron and Janig, 1991). These preganglionic sympathetic neurons send their

axons to sympathetic ganglion cells of the bladder in the sympathetic chain (T12

– L6), pelvic ganglia, and to the inferior mesenteric ganglia (Vera and Nadelhaft,

1992). The caudal continuation of the inferior mesenteric ganglion forms the

hypogastric nerve, which carries the majority of the sympathetic input to the pelvic plexus. A small number of hypogastric fibers have their cells of origin

located in either the sympathetic chain or the inferior mesenteric ganglion.

The pelvic nerve carries the parasympathetic input and originates from the

last lumbar (L6) and first sacral (S1) spinal nerves in the rat (Purinton et al.,

1973). The pelvic nerve also carries a small portion of sympathetic fibers to the

pelvic viscera. Numerous small efferents arise from the pelvic ganglion and

supply the rectum, the ureter, the vas deferens, the seminal vesicle, the prostate,

the bladder, and the urethra. In the female rat the pelvic ganglion, which is also

referred to as the paracervical or Frankenhauser ganglion, is smaller than that in

males and lies against the lateral wall of the uterine cervix. Afferent neurons with

fibers in the pelvic nerve are located in the L6-S1 dorsal root ganglia in the rat

and carry sensory information from the descending colon, bladder, urethra and

sex organs (Nadelhaft and Booth, 1984). These fibers enter the tract of Lissauer,

encircle the dorsal horn with a medial component terminating in the dorsal

commissural gray and the lateral component terminating in the sacral

parasympathetic nucleus.

The pudendal nerve arises from L5-L6 in the rat and carries efferent fibers

to the coccygeus, internal obturator, ventral and dorsal bulbospongiosus (also

termed bulbocavernosus), ischiocavernosus, external urethral and anal sphincter muscles, and afferent fibers from the penis, prepuce, scrotum, and ventral- proximal tail (McKenna and Nadelhaft, 1986). The sacral plexus is the complex formed by the bridge-like structure connecting the pudendal nerve with the lumbosacral trunk, and two nerve branches emerging from it, one innervating the proximal half of the scrotal skin, and the other, known as the motor branch, innervating the muscles at the base of the penis (Pacheco et al., 1997). These branches are only considered as a part of the sacral plexus because they integrate axons from both the lumbosacral trunk and pudendal nerve.

In rats, retrograde tracer studies of the pudendal nerve have demonstrated the presence of two motor nuclei in the ventral horn gray matter of the L5-L6 spinal cord segments, the dorsomedial and dorsolateral cell columns

(Figure 2.1; Breedlove and Arnold, 1980; McKenna and Nadelhaft, 1986; Vera and Nadelhaft, 1992). These nuclei are the homologues to the nucleus of Onuf, which is found in other species, and contains motor neurons innervating the anal and urethral sphincters, bulbospongiosus and ischiocavernosus muscles, all located in one cell group. McKenna and Nadelhaft (McKenna and Nadelhaft,

1986) also demonstrated that in the female rat, urethral sphincter motor neurons accounted for almost all the dorsolateral nucleus motor neurons, and anal sphincter motor neurons accounted for almost all the dorsomedial motor neurons; the ischiocavernosus and bulbospongiosus muscles are vestigial in the female rat. In the male, neurons innervating the external anal sphincter and bulbospongiosus muscles are found intermingled in the dorsomedial nucleus. In

contrast, in the dorsolateral nucleus, the urethral sphincter neurons are located in

the lateral portion of the nucleus and the ischiocavernosus neurons are located in

the medial portion (Collins et al., 1992; Hermann et al., 1998; McKenna and

Nadelhaft, 1986). There are significantly more neurons in both nuclei in the male than in the female and the neurons are larger in the male. However, perineal muscle tracer injections have established that there is no difference between males and females in the number of motor neurons innervating the external anal or urethral sphincters (McKenna and Nadelhaft, 1986).

Figure 2.1 A-F: The caudal lumbar and rostral sacral spinal cord contains the neuronal circuitry that organizes the parasympathetic components of bladder, bowel and sexual function. The following abbreviations are used in this figure: DCG: dorsal commissural gray; SPN: sacral parasympathetic nucleus; DL: dorsolateral nucleus; DM: dorsomedial nucleus; DF: dorsal funiculus; DH: dorsal horn; CC: central canal; VM: ventromedial nucleus; RDL: retrodorsolateral nucleus. A. The cord at this level contains the SPN and the associated somatic motor columns innervating the pelvic floor musculature, the DM, and DL nuclei. B,C. The L6 segment contains the DM and DL nuclei that are characterized by a lack of myelinated fibers; this is most easily seen in the DL. The boxed area in B is shown at higher magnification in C. In this particular section, bulbospongiosus motor neuron cell bodies are retrogradely labeled with horseradish peroxidase (HRP) and can be easily identified in the higher power image shown in C. This is a plastic embedded section containing the labeled bulbospongiosus motor neurons cell bodies; diaminobenzidine was used to visualize the horseradish peroxidase in the neurons. After processing the tissue for HRP, it was stained en bloc with osmium and uranyl acetate and embedded in plastic for subsequent sectioning for electron microscopy. Myelinated fibers appears black (note the darker staining of the ventral white commissure just above the DM nuclei and in the white matter of the dorsal lateral and ventral funiculi). The retrogradely labeled neurons also appear dark in this section (arrow in C). D. This micrograph shows a 1.0 μm thick, toluidine blue stained section of the

Continued

Figure 2.1 continued

DM area enclosed by the circle in C. Two motor neuron cell bodies are shown in cross section; the cell on the left has a clear nucleus, clumps of rough endoplasmic reticulum in the cytoplasm and a large primary dendrite extending dorsally (toward the letter D). The other cell body has clumps of rough endoplasmic reticulum and short primary dendrites that extend mostly out of the plane of section. There is a high density of cross-cut dendrites which appear as small white circles; these are rostrocaudally oriented bundles of dendrites. Black myelinated fibers can be seen at the top of the micrograph and a couple fibers can be seen to traverse the nucleus between the two cell bodies. The magnification is too low to identify the HRP stained elements in the cytoplasm. E,F. These low power dark field micrographs of adjacent sections show the extensive dendritic arborizations of bulbospongiosus motor neurons retrogradely labeled with horseradish peroxidase from muscle injections (Tetramethyl benzidine processing). The bundles extend into the DCG region that receives pudendal and pelvic nerve afferent input, into the DL nucleus that contains motor neurons of the ischiocavernosus and external urethral sphincter muscles, and over to the contralateral DM nucleus. (From: Nout, Y. S. et al., 2005a).

These nuclei have a number of special anatomic features (Figure 2.1) similar to

the phrenic nucleus, a motor nucleus in the cervical spinal cord that innervates

the diaphragm. These nuclei lack myelinated fibers making them appear

translucent, similar to the substantia gelatinosa. The motor neurons exhibit

extensive dendritic bundling (McKenna and Nadelhaft, 1986; Roney et al., 1979;

Sasaki, 1994; Schroder, 1980); the rostrocaudally oriented dendritic bundles can

be seen in Figure 1D. Dense bundles of dendrites from bulbospongiosus motor

neurons can be seen in Figure 1E and F (arrows), interconnecting the

dorsomedial nuclei on the two sides as well as the dorsomedial and dorsolateral nuclei on the same side. A dorsally extending group projects into the dorsal commissural gray (Figure 1E&F). Peshori et al. (1995) demonstrated that motor neurons in the contralateral dorsomedial nucleus could be transneuronally labeled with wheatgerm-agglutinin horseradish peroxidase after a unilateral muscle injection and dye-coupling has been demonstrated between bulbocavernosus motor neurons by Matsumoto et al. (Matsumoto et al., 1988).

At the ultrastructural level, the dendrites are connected with tight junctions or puncta adherentia (Nout, Y. S. et al., 2005a; Ramirez-Leon and Ulfhake, 1993) as well as through presynaptic dendrites (Nout, Y. S. et al., 2005a). The synaptic arrangements in this region are also somewhat unique in that axon terminals contacting more than one dendrite is common ((Nout, Y. S. et al., 2005a;

Ramirez-Leon and Ulfhake, 1993). The dense innervation of this region by γ- amino butyric acid (GABA)-containing elements is also of note (Li et al., 1995;

Ramirez-Leon and Ulfhake, 1993). It is likely that these anatomical arrangements

can provide for a more synchronized activation of motor neurons controlling

pelvic floor musculature (e.g. the sphincters and erectile musculature).

The pudendal motor neuron dendrites occupy most of the ventral horn and

reach dorsally beyond the central canal (Hermann et al., 1998; Peshori et al.,

1995; Sasaki, 1994). In the cat, the sphincter motor neuron dendritic arbors have

been shown to extend into the regions where functionally associated

preganglionic neurons in the sacral parasympathetic nucleus are located (Beattie et al., 1990). For example, the external anal sphincter motor neurons project dendrites into the dorsal band of the sacral parasympathetic nucleus where the colonic preganglionics are located and the external urethral sphincter motor neurons into its lateral band where the bladder preganglionics reside. Similarly, the bulbospongiosus motor neurons in the rat extend profuse dendrites into the dorsal commissural region (Figure 1E) where interneurons important in sexual reflex function are located (Nadelhaft and Booth, 1984; Peshori et al., 1995;

Nadelhaft and Vera, 2001).

Pudendal nerve afferent neurons are located in the L6 and S1 dorsal root

ganglia (Nadelhaft and Booth, 1984). In both sexes, pudendal afferent fibers in

the spinal cord are located in the dorsal columns, the medial half of Lissauer's

tract, the extreme medial edge of the dorsal horn, both ipsilaterally and

contralaterally, and in a large terminal field in the dorsal gray commissure. No

afferents have been detected in the intermediate or ventral gray (McKenna and

Nadelhaft, 1986).

Supraspinal control mechanisms

Although in the rat eliminative and sexual reflex patterns are mainly organized at the spinal level, there is evidence for supraspinal centers that may

modify the functional response. These supraspinal inputs strongly appear to

influence the magnitude and coordination of these reflexes, and generally this

descending supraspinal input is inhibitory to the lumbosacral reflexes.

Retrograde tracer injections into the ventromedial gray of the lumbar spinal cord

labeled supraspinal input primarily from the vestibular nuclei, the gigantocellular reticular nuclei, the medullary raphe nuclei, and the hypothalamic paraventricular

nuclei (Marson et al., 1992; Marson and McKenna, 1990; Monaghan and

Breedlove, 1991; Shen et al., 1990). The ventrolateral gray of the lumbar spinal

cord has supraspinal input from the dorsolateral pontine tegmental region, also referred to as Barrington’s nucleus or the pontine micturition center (Ding et al.,

1995). Furthermore, transneuronal tracing studies using pseudorabies virus

injections directly into the muscles involved in pelvic floor reflexes have

consistently confirmed these same regions to be the source of supraspinal input

(Marson and McKenna, 1996; Tang et al., 1999; Vizzard et al., 2000). Some studies have focused on supraspinal input originating from the nucleus raphe obscurus in the brainstem and the region in the ventrolateral medulla referred to as the gigantocellular – lateral paragigantocellular complex. Projections from this latter area are widespread and include fibers in close apposition to external anal sphincter and bulbospongiosus motor neurons, and although targets from the nucleus raphe obscurus are much more restricted, they also include contacts 17

between nucleus raphe obscurus terminals and bulbospongiosus and external

anal sphincter motor neurons (Hermann et al., 1998; Hermann et al., 2003). More

specifically, fibers from the nucleus raphe obscurus ramify in close apposition to

both bulbospongiosus and external anal sphincter motor neurons and terminals

of nucleus raphe obscurus projections in the lumbosacral spinal cord have been

shown to co-localize with serotonin, thyrotropin releasing hormone, and

substance P (Ramirez-Leon et al., 1994).

In fact, the nucleus raphe obscurus is thought to be an overall regulator of

autonomic functions controlled by both cranial and spinal autonomic efferents.

Rostral portions of the nucleus raphe obscurus project to the dorsal vagal complex, which controls proximal digestive functions (McCann et al., 1989), whereas caudal portions of the nucleus raphe obscurus project to the caudal intermediolateral cell column, sacral parasympathetic nucleus, and related somatic motor neurons, which control distal digestive and other pelvic autonomic functions. Also, the nucleus raphe obscurus maintains direct projections to both autonomic and somatic regions and to regions of the intermediate gray, which contain putative interneurons that contribute to the organization of pelvic floor reflexes. The nucleus raphe obscurus is thus in a position to modulate autonomic preganglionic and functionally related skeletal motor neuron activity (Hermann et al., 1998).

Electrical stimulation of the nucleus raphe obscurus results in reduction of

spontaneous anorectal activity, providing evidence for a direct brainstem

inhibitory circuit (Holmes et al., 1997a). Also, nucleus raphe obscurus lesions 18

elicit transient increases in anorectal reflex activity (Beattie et al., 1996; Holmes et al., 2002) and disrupt male copulatory behavior in rats (Yamanouchi and

Kakeyama, 1992). However, nucleus raphe obscurus lesions do not have an effect on measures of ex copulatory penile reflexes (Holmes et al., 2002). In contrast, lesions of the gigantocellular – lateral paragigantocellular complex did not affect measures of external anal sphincter activity but altered penile reflexes significantly (Holmes et al., 2002). It has been shown that the bulbospongiosus muscle displays electromyographic activity during the passage of urine (Sachs and Leipheimer, 1988; Schmidt, M. H. et al., 1995), and it is suggested that the nucleus raphe obscurus projections may be specific to pudendal eliminative reflexes, while other descending brainstem projections are specific to sexual reflexes (Holmes et al., 2002).

Projections from the gigantocellular – lateral paragigantocellular complex

are diffuse, descending to all levels of the spinal cord affecting sensory, motor,

and autonomic control circuits beyond those for pudendal reflexes. The

gigantocellular – lateral paragigantocellular complex may globally modulate multiple behaviors with somatic and autonomic components such as reproductive

reflexes, acoustic startle, and nociception as proposed by (Hermann et al., 2003)

Neurotransmitters implicated in the descending supraspinal control of

pelvic and pudendal motor neurons include serotonin, thyrotropin-releasing

hormone, and GABA. Medullary thyrotropin-releasing hormone neurons diffusely

project to the ventral horn of the spinal cord and sympathetic neurons within the

intermediolateral cell column (Appel et al., 1987; Hirsch and Helke, 1988). When

applied intrathecally at the level of the L5-L6 spinal cord, thyrotropin-releasing

hormone resulted in contraction of the internal anal sphincter, predominantly

through activation of the pelvic nerve. Although sectioning of the hypogastric

nerve had no effect on thyrotropin-releasing hormone-induced internal anal

sphincter activity, a role for thyrotropin-releasing hormone activation of sympathetic preganglionic neurons should not be excluded since the pelvic nerve has been shown to contain both sympathetic and parasympathetic fibers

(Hulsebosch and Coggeshall, 1982). It is suggested that the relative roles of the pelvic and hypogastric nerves in internal anal sphincter contractility differ with changes in activity of rectal afferents (Holmes et al., 1995). Also, pudendal motor neurons are under spinal descending inhibitory control. Similar to the effect thyrotropin-releasing hormone has on the internal anal sphincter, high doses of thyrotropin-releasing hormone applied to the lumbosacral spinal cord resulted in an increase in firing rate of the external anal sphincter (Holmes et al., 1997b).

Thus, thyrotropin-releasing hormone appears to be involved in maintaining fecal continence. Other studies have shown that thyrotropin-releasing hormone plays a critical role in modulation of gastrointestinal function (McCann et al., 1989).

Thyrotropin-releasing hormone has been shown to increase gastric secretion and motility, intestinal transport, and is thought to play a role in stress-induced increases in defecation (Miyata et al., 1992). Although thyrotropin-releasing hormone appears to have an excitatory effect on external anal sphincter motor neurons, intrathecally applied thyrotropin-releasing hormone has an inhibitory effect on penile erections, suggesting that although external anal sphincter and

bulbospongiosus motor neurons are co-mingled within the same spinal nucleus,

they are discretely and differentially regulated by separate neural circuits

(Holmes et al., 1997b).

Eliminative functions following spinal cord injury

Following acute injury to the spinal cord an initial period of “spinal shock”

occurs, that is characterized by areflexia and generally lasts for approximately 24

hours in humans (Ditunno et al., 2004). Disruption of spinal cord tracts proximal to the lumbosacral cord, leads to loss of supraspinal control over normal eliminative and reproductive behaviors. These functions, which reflect coordinated activity of both somatic and autonomic components of the nervous system, are important therapeutic targets as they represent critical problems for the spinal cord injured population, and have been less well studied than locomotion in models of SCI (Anderson, 2004). After the initial post injury period of spinal shock the basic spinal reflexes control these functions independent from supraspinal input. During the recovery phase from spinal shock (1 day – 12 months) a gradual return of reflexes may take place as well as the development of hyperreflexia and spasticity (Ditunno et al., 2004). Interestingly in the rat, the initial loss and subsequent recovery of functions that have autonomic components show a similar pattern of recovery as locomotor function (Holmes et al., 2005) suggesting commonality of underlying recovery mechanisms.

In rat models, in addition to loss of control of eliminative and sexual function, the lumbosacral reflexes develop hyper-reactivity, including increased

external anal sphincter contractions after distention and hyper-reflexia of

erections in response to slight tactile stimuli (Holmes et al., 1998; Holmes et al.,

2005). This is consistent with the finding in humans, in which removal of

supraspinal control leads to spasticity and dyssynergia of urethral and anal

sphincters. In addition to hyperreflexia of the external anal sphincter, it is well

recognized that hyperreflexia of the detrusor occurs after SCI, as well as

detrusor-sphincter-dyssynergia.

We suggest that in addition to these muscles, other pelvic floor muscles,

and specifically the bulbospongiosus musculature develops hyperreflexia and

spasticity following SCI. This change in reflex activity is reminiscent of

hyperreflexia observed in other segmentally mediated reflexes (Bose et al.,

2002). Interestingly, McKenna et al. (McKenna et al., 1991), observed simultaneous contraction of all pelvic floor musculature (bulbospongiosus, ischiocavernosus, external anal sphincter and external urethral sphincter) during

the urethrogenital reflex after spinal cord transection. This reflex is induced by stimulation of the urethra and these authors suggest that the motor program is controlled by a spinal pattern generator in the absence of descending input and that the spinal circuitry is responsible for the rhythmic, coordinated bursting activity of all these muscles. Certainly, the extensive co-mingling of the dendrites of these motor neurons in the spinal cord (as well as their electrotonic and synaptic coupling) could be an anatomical substrate for this and other coordinated activities (Coolen et al., 2004; Giuliano and Rampin, 2004).

A closer examination of the bulbospongiosus muscle activity after

contusion injury of the spinal cord (Nout et al., 2005) in awake behaving rats,

shows that the loss of descending input produces an exaggerated contraction

pattern in this muscle similar to that seen in the external anal sphincter. This

activity was observed during erectile events as well as during micturition. Using

pressure recordings from the corpus spongiosum penis it is possible to reliably

assess both micturition and erectile events (Nout, Y.S. et al., 2004; Schmidt,

M.H. et al., 2004). Following spinal cord damage, increased corpus spongiosum penis mean pressures and increased duration of micturition as well as of erectile events occur. Increased corpus spongiosum penis pressures indicate bulbospongiosus muscle spasticity, since pressure within the bulb of the corpus spongiosum penis is directly dependent on the activity in the surrounding bulbospongiosus musculature (Schmidt, M. H. et al., 1995).

In addition to loss of descending inhibitory control of spinal reflex

pathways, plasticity within the spinal cord is thought to contribute to recovery but

also to development of exaggerated spinal reflex responses. For example,

Weaver and her colleagues have shown that sprouting of dorsal root afferents

containing calcitonin gene-related peptide may contribute to the development of

autonomic dysreflexia after severe SCI (Weaver et al., 2001). Similarly, calcitonin

gene-related peptide fibers contributing to the parasympathetic components of

bladder, bowel, and sexual reflex function have also been demonstrated to sprout after SCI in cats (Beattie et al., 2000b). Spinal cord injury, in this species, produces a chronic (measured at 6 weeks) denervation of identified parasympathetic preganglionic neurons (Beattie et al., 1993); the proportion of 23

the somatic and proximal dendritic membrane of these efferent neurons

contacted by synaptic terminals is significantly reduced, the size of the terminals is smaller, and glial coverage is reciprocally increased. In additional studies, an

increase in the proportion of the membrane area contacted by calcitonin gene-

related peptide immunoreactive terminals appears to increase (Beattie et al.,

2000) suggesting that the effect of such input might be even more functionally

significant after SCI. Interestingly, the synaptic inputs to Onuf’s nucleus in the

same studies, showed similar synaptic rearrangements, i.e. reduced size of

terminals apposed to Onuf’s motor neurons after SCI (Beattie et al., 1993;

Behrmann et al., 1993), and an increase in their terminal coverage by GABA

immunoreactive terminals (Beattie et al., 2000b). The plasticity that is seen in this

area of the cord forms an important target for potential therapeutic interference.

For example, it has been shown that interference in the development of the

primary afferent plasticity affected the development of autonomic dysreflexia

(Weaver et al., 2001) and detrusor-sphincter-dyssynergia (Seki et al., 2004).

Recently, Cameron et al. (Cameron et al., 2004) presented a strategy for modulation of post-traumatic spinal plasticity in both sacral afferents and propriospinal projection neurons via targeted gene therapy in a model of autonomic dysreflexia.

2.2 Micturition

When compared to defecatory and sexual function, urinary function has

been studied most extensively in animal models of SCI. Upper urinary tract 24

function relates to the production of urine in the kidneys and continuous supply of urine into the bladder through two ureters. Lower urinary tract function involves storage and complete controlled emptying of urine from the bladder. The lower urinary tract is comprised of the bladder, internal and external sphincters, and urethra. The smooth muscle in the bladder wall is termed the detrusor muscle.

Detrusor muscle contraction is mediated primarily by parasympathetic stimulation. Parasympathetic control of the detrusor muscle originates from the preganglionic axons in the pelvic nerve. The hypogastric nerve supplies the sympathetic input to the detrusor muscle. Sensory information from the bladder wall travels within the pelvic nerve to the spinal cord. The internal sphincter is located at the junction of the bladder and urethra and is, similar to the bladder wall, also comprised of smooth muscle. Similar to the detrusor muscle, the internal sphincter receives sympathetic and parasympathetic innervation; however, in the internal sphincter sympathetic stimulation causes contraction.

The external sphincter is comprised of striated muscle that surrounds the urethra and is innervated through the pudendal nerve.

The storage function of the bladder is mediated predominantly through two components of the sympathetic innervation. The first is closure of the bladder outlet through contraction of the bladder neck and internal sphincter which is mediated through α-adrenergic receptors. The second is relaxation of the bladder wall which is mediated through β-adrenergic receptors. Under normal circumstances filling of the bladder occurs with little increase in bladder pressure.

As bladder filling increases, sensory input from the bladder wall to the sacral

spinal neurons increases until the threshold for the micturition reflex is reached.

For voiding the bladder, a coordinated process is required that involves detrusor

muscle contraction in combination with relaxation of the internal and external

sphincters, urethra, and pelvic floor musculature. Under normal circumstances this process is under voluntary control and occurs through well-regulated and integrated autonomic and somatic reflexes, but after SCI changes occur at the

level of the bladder and spinal cord that result in malfunction of the lower urinary

tract.

The complete loss of descending input initially causes an absence of

bladder emptying which is gradually replaced by spinal reflex circuit activity that

is initiated by a different population of afferents (e.g. c-fiber afferents) than in the

normal condition (de Groat et al., 1998; de Groat et al., 1990; de Groat and

Yoshimura, 2005). In most cases, reflex detrusor activity reappears 2 – 12 weeks

after the spinal shock phase (Chancellor and Blaivas, 1996; Fam and Yalla,

1988). The effect of incomplete SCI on lower urinary tract function depends on

the severity of the primary injury (Wrathall and Emch, 2005). While some

recovery of this function occurs, residual deficits remain in the coordination of the

autonomic efferents to the detrusor muscle and the somatic efferents to the

external urethral sphincter muscle (so-called detrusor-sphincter-dyssynergia)

producing inadequate bladder emptying. When the supraspinal, coordinated

control of these systems is lost, the resulting bladder dysfunction is termed

“neurogenic bladder impairment” or a “neurogenic bladder” (Potter, 2005).

Detrusor-sphincter-dyssynergia is thought to be the main reason for the

observed increased voiding pressures and decreased voiding efficiency, and has

been documented in rats (Cheng and de Groat, 2004; Kruse et al., 1993; Pikov et al., 1998; Yoshiyama et al., 2000). Although external urethral sphincter bursting activity can be mediated by spinal reflex mechanisms, the bursting activity that is seen after SCI is abnormal, leading to shorter urethral opening times and this presumably contributes to the inefficient voiding and increased voiding pressures

(Cheng and de Groat, 2004). Also, the amplitude from external urethral sphincter electromyographic recordings has been shown to increase following SCI (Pikov and Wrathall, 2001). Suppression of external urethral sphincter activity results in improvement of all voiding parameters (Kruse et al., 1993; Yoshiyama et al.,

2000). In addition to dyssynergia, detrusor hyperreflexia has been well characterized in rats following SCI (Mitsui et al., 2003) and is thought to be due to a lack of supraspinal inhibition with or without an increase of afferent signaling.

Moreover, enlargement of the bladder may result in plasticity of afferents, further

contributing to this hyperreflexia (de Groat et al., 1998). The significance of this syndrome in the recovery of micturition, however, is unknown. Recovery of micturition function has been observed to occur through amelioration of detrusor- sphincter-dyssynergia without noticeable change of detrusor hyperreflexia (Mitsui et al., 2003). The authors of this report suggest that the improved external urethral sphincter function and lack of concomitant improved detrusor muscle

function, reflects comparatively easier recovery of somatic nerves vs. autonomic

nerves (Mitsui et al., 2003).

Studies in animals indicate that recovery of bladder function after SCI is

dependent on reorganization of reflex pathways in both the peripheral and central

nervous systems. The ultimate goal of research is restoration of normal function,

however providing better management of the neurogenic bladder is an important

objective.

2.3 Defecatory function

Defecatory function after SCI has been least studied compared to

micturition and sexual function, however, exhibits similar responses to the loss of descending control causing inadequate voiding, fecal impaction and chronic constipation in man (Cosman et al., 1991; Frenckner, 1975) due, perhaps, to dyssynergia and external anal sphincter spasticity (Holmes, 2005; Holmes et al.,

1998).

The most distal portion of the gastrointestinal tract is formed by the rectum

and anus. Innervation of the rectum is similar to that of the colon, but innervation

of the anus is more complex (for a comprehensive review, see Janig and

McLachlan, 1987). The anus controls passage of fecal matter by the degree of

constriction of the internal and external anal sphincters (Gonella et al., 1987;

Schuster, 1968). The internal anal sphincter is formed by thickening of circular smooth muscle that lies immediately inside the anus. Similar to the rest of the gastrointestinal smooth muscle, the internal anal sphincter contains a myenteric plexus within the tunica muscularis. Furthermore, the internal sphincter muscle receives autonomic innervation via the pelvic plexus. The external anal sphincter is composed of striated muscle that surrounds the internal anal sphincter and 28

extends distal to it. The external sphincter is controlled by nerve fibers in the

pudendal nerve, which in the rat has motor, sensory and autonomic components

(Holstege and Tan, 1987; Katagiri et al., 1986; Paxinos, 1995; Rexed, 1954).

While the internal anal sphincter is the main mechanism for continence,

accounting for the majority of the resting pressure, subconsciously the external

sphincter is usually kept constricted unless conscious signals inhibit constriction

(Gonella et al., 1987). Motor units of the external anal sphincter have been

shown to be tonically active (Gonella et al., 1987; Krier, 1985), whereas the other

pudendal motor units are effectively quiescent (Holmes et al., 1994).

The rectum and anal canal are supported by the pelvic diaphragmatic

musculature, which participates in the functions of fecal continence and defecation. Paired rectococcygeal muscles originate on each side of the rectum

and attach dorsally to the base of the tail. These smooth muscles are innervated

by autonomic fibers from the pelvic plexus and shorten the rectum during

defecation to assist evacuation of feces. Laterally the illeocaudalis and

pubocaudalis and the coccygeus muscles surround the rectum. These striated

muscles are innervated by the L6 to S1 nerves in the rat (Bremer et al., 2003)

and help to compress the rectum during defecation.

In normal animals and humans, defecation is initiated by defecation

reflexes (Gonella et al., 1987; Guyton and Hall, 2000). One of these reflexes is

an intrinsic reflex mediated by the local enteric nervous system in the rectal wall.

Distention of the rectal wall initiates afferent signals that spread through the

myenteric plexus to initiate peristaltic waves in the descending colon, sigmoid, 29

and rectum, forcing feces toward the anus. As the peristaltic wave approaches the anus, the internal anal sphincter is relaxed by inhibitory signals from the

myenteric plexus. A rectoanal inhibition reflex occurs in which as the rectum fills and the internal anal sphincter relaxes, the external anal sphincter contracts involuntarily and defecation only occurs following further rectal distension inducing concomitant external anal sphincter relaxation (Gonella et al., 1987).

This local intrinsic defecation reflex, however, is by itself relatively weak and usually must be fortified by another type of defecation reflex, a parasympathetic defecation reflex that involves the sacral segments of the spinal cord. When

nerve endings in the rectum are stimulated by distension, signals are transmitted

first into the spinal cord and then reflexively back to the descending colon,

rectum, and anus by way of parasympathetic nerve fibers in the pelvic nerves.

These parasympathetic signals greatly intensify the peristaltic waves and relax

the internal anal sphincter and thus convert the intrinsic myenteric defecation reflex from a weak effort into a powerful process of defecation.

The afferent defecation signals entering the spinal cord initiate other effects such as closure of the glottis and contracture of the abdominal wall

muscles to force fecal contents of the colon downward and at the same time

cause the pelvic floor to relax downward and pull outward on the anal ring to

evaginate feces. In the rat reflex defecation is mainly organized at the spinal level but there is evidence for supraspinal centers that may modify the functional response (Maggi et al., 1988). These are described in more detail in the general section of this chapter.

In human patients with SCI, loss of input from proximal to the lesion to the bowel results in reduced transit through the bowel. Additionally, coordination of autonomic and somatic defecation reflexes is lost secondary to disruption of supraspinal control pathways. Compressive destruction of the conus medullaris of the spinal cord can destroy the distal segments of the cord where, in humans, the cord defecation reflex is integrated, and this almost always paralyzes defecation. More frequently the spinal cord is injured more proximal, between the conus medullaris and the brain, in which case the voluntary portion of the defecation act is blocked while the basic spinal cord reflex for defecation is still intact. As described above, there is evidence for development of increased anal sphincter resting pressure or hyperreflexia after SCI (Holmes et al., 1998;

Holmes et al., 2005; Sun et al., 1995). A greater understanding of the role of pelvic reflexes in controlling evacuation and continence is required for development of neuromodulation strategies to improve quality of life after SCI

(Chung and Emmanuel, 2005; Lynch and Frizelle, 2005).

2.4 Sexual function

Spinal cord injury has an enormous impact upon sexual function of both men and women. Male fertility and sexual function are greatly impaired by SCI with less than 5% of men able to procreate without medical intervention (Talbot,

1955; Thomas, 1983). In women laboratory studies have shown that 44% of women with SCI were able to achieve orgasms compared to 100% of able- bodied controls (Sipski et al., 2001). Regaining sexual function is the highest 31

priority for paraplegics as demonstrated in a recent survey (Anderson, 2004).

Our understanding of the complex neural pathways and mechanisms

underlying sexual function and behavior is limited, but sexual function requires

participation of autonomic and somatic nerves and integration with two

interconnected portions of the spinal cord, which in turn make connections with

the brainstem and cerebral cortex (Giuliano and Rampin, 2000, 2004; Hubscher,

2005; McKenna, 2000; Steers et al., 1988). Similarly as described for micturition

and defecation function, the main peripheral nerves involved in sexual function

are the pelvic, hypogastric, and pudendal nerves.

Penile erection is a vascular event associated with tumescence of the

cavernous bodies and is only a single component of the complex sexual

behavior. For a detailed review see (Giuliano and Rampin, 2000, 2004). In

humans, erection occurs in response to tactile, visual, and imaginative stimuli. In

animals olfactory and auditory cues are also important stimuli. Parasympathetic innervation, through the pelvic nerve, provides the major excitatory input, responsible for vasodilation of the penile vasculature and erection. Sacral preganglionic fibers travel to the pelvic plexus in the pelvic nerve. The cavernous nerve exits the pelvic plexus and innervates different penile structures. Since the pelvic plexus receives sympathetic input as well, the cavernous nerve contains parasympathetic and sympathetic fibers, providing the vasodilator and vasoconstrictor input to penile smooth muscle. Although the sympathetic nervous system appears to play a role in detumescence of the penis, this pathway may facilitate erections after injury to parasympathetic pathways. Sympathetic 32

preganglionic nerve fibers to the penis originate from neurons in the

intermediolateral cell column and intercalated nucleus of the T9-L2 spinal cord

segments. These preganglionic neurons receive input from supraspinal sites and pelvic viscera. Sympathetic axons travel to the penis through the pelvic, cavernous and pudendal nerves.

The external genitalia and perineum are innervated by somatic myelinated

and unmyelinated afferent fibers. These fibers convey information to the lower

lumbar and upper sacral segments of the spinal cord via the pudendal nerve and

via branches of the pelvic, genitofemoral, ilioinguinal, and anococcygeal nerves

(Hubscher, 2005). These autonomic afferents travel in the dorsal nerve of the

penis, which is one of the branches of the pudendal nerve. Afferent input from

the penile skin, prepuce, and glans conveyed by the dorsal nerve of the penis

initiates and maintains reflexogenic erections.

The reproductive structures innervated by the hypogastric nerve are

involved in contraction and movement of substances. The hypogastric nerve is

believed to play a role in sperm motility and ejaculation. Sectioning of this nerve

does not have an effect on mating behavior (Hubscher, 2005). For a detailed

review on regulation of ejaculation see (Coolen et al., 2004).

The sacral spinal cord is essential for penile erection. Destruction of the

sacral spinal cord or its outflow, including the conus medullaris, abolishes

erections. Reflexive erections are mediated by a sacral spinal reflex with a

sensory input through the dorsal nerve of the penis, and motor pathways

consisting of sacral parasympathetic nerves conveyed by the pelvic and eventually cavernous nerves. Supraspinal excitatory and inhibitory mechanisms within the brain and spinal cord coordinate and integrate these neural inputs. In the cat and rat, somatic afferents from the pudendal and cavernous nerves terminate in the medial spinal cord (laminae III and IV) and dorsal commissure

(lamina X) of the sacral spinal cord. Afferents in the cavernous nerve terminate in

Lissauer’s tract (lamina I), the lateral and medial collateral pathways and just dorsal to the parasympathetic preganglionic nucleus.

After SCI, sexual reflexes are disrupted similar to micturition and defecatory reflexes. Two distinct control mechanisms induce penile erections in humans: reflexogenic and psychogenic. Spinal cord injury above the sacral level will not only preserve this reflex but can even enhance it, especially if the lesion is complete, such as seen in priapism (Elliott, 2005). The ability to have reflex erections is lost if the sacral spinal cord is injured or if the pudendal and / or pelvic nerves are damaged (Elliott, 2005). Psychogenic erections occur in men with an intact nervous system in response to various stimuli and men with injuries to their sacral spinal cord are dependent on intact psychogenic pathways for erectile activity. Reflexogenic and psychogenic mechanisms probably act synergistically to determine erectile response. Although 95% of men with complete spinal cord injury and upper motor neuron lesions experience penile erections, the erection is often of poor quality or poorly sustained (Brown et al.,

2005). Clinical literature has indicated that thoracolumbar contusion produces transient priapism following tactile stimulation, suggestive of the presence of 34

descending inhibitory pathways that normally suppress this segmental excitatory reflex. In humans, loss of descending input results in facilitated spinally mediated reflex erectile activity but disruption of ejaculatory function and seminal fluid composition (Basu et al., 2004; Higgins, 1979). Although currently an erection can be provided through safe and effective erection enhancement methods, neither ejaculatory dysfunction nor penile sensation and orgasm can be reversed

(Elliott, 2005).

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CHAPTER 3

A NOVEL TECHNIQUE FOR MONITORING MICTURITION AND SEXUAL

FUNCTION IN MALE RATS USING TELEMETRY

3.1 Introduction

In animal models of spinal cord injury (SCI) emphasis in general lies on

examination of locomotor function. Outcome measurements examining various

aspects of locomotor function exist in large numbers. However, in humans, impairment of lower urinary tract function following SCI and its subsequent complications are highly prevalent and clinically very important (Anderson, 2004;

Noreau et al., 2000; Schmidt and Schmidt 2004). The state of urologic function is critical and continued investigation of pathophysiology and treatments to improve recovery of this aspect of autonomic function are, therefore, essential.

Existing methods to examine micturition function in rats include

cystometric and electromyographic (EMG) techniques (Conte et al., 1988; Kruse

et al., 1993; Pikov et al., 1998; Pikov and Wrathall, 2001) and monitoring animals

in metabolic cages (Haas et al., 1997; Sakamoto et al., 2000; Schmidt, F. et al.,

2001). Limitations of these techniques are use of anesthesia or heavy sedation and placement of catheters in bladder or urethra, both of which are known to 42

alter the micturition reflex (Cannon and Damaser, 2001; Conte et al., 1988;

Yoshiyama et al., 1999). Furthermore, animal well-being may be in jeopardy when animals are placed on a wire mesh surface in metabolic cages for extended periods of time, particularly when animals are paralyzed. Existing methods to study sexual function in rats include animal observation in either copulatory (Hart, 1968) or non-copulatory (Sachs et al., 1994) settings and ex copulatory reflex erection tests (Holmes et al., 1988; Sachs, 1985). Limitations of these tests include difficulty in accurately recognizing various aspects of rat sexual behavior and non-physiologic testing situations.

Investigators of penile erection physiology have designed a technique to

monitor erectile events in awake rats by continuously recording pressures within

the corpus cavernosum of the penis (CCP) (Giuliano et al., 1994) or the corpus

spongiosum of the penis (CSP) (Schmidt, M. H. et al., 1994). During erections

blood enters the cavernous sinuses of the CCP and CSP causing expansion of the spongy erectile tissue and the penis to become engorged and erect. The exact roles that the ischiocavernosus (IC) and bulbospongiosus (BS) muscles, two muscles of the pelvic floor that lie over the CCP and CSP respectively, play in this process is not fully elucidated; however both of these muscles have been shown to display electromyographic (EMG) activity during visually confirmed erections (Beckett et al., 1973; Beckett et al., 1975; Holmes et al., 1991; Holmes and Sachs, 1992; Schmidt, M. H. et al., 1995) as shown in Figure 3.1.

Figure 3.1: Electromyographic activity of the BS and IC muscles during erectile events. Examples of erectile events involving several glans erections and flips of the penile body are shown. Note that the entire erectile event is associated with an increase in baseline CSP pressure. Glans erections (E1, E2, E3) were directly associated with BS muscle bursts and flips of the penile body (arrowheads) were associated with IC muscle bursts. CSP = corpus spongiosum penis; BS = bulbospongiosus muscle; IC = ischiocavernosus muscle; EMG = electromyogram (From: Schmidt et al., 1995)

Contraction of the IC muscles is considered critical for the development of penile

rigidity and contraction of the BS muscle is considered essential for ejaculation.

To what extent the BS muscles are involved in penile rigidity, and what the role of

BS muscle contraction is during non-ejaculatory erections is not clear (Schmidt,

M. H. and Schmidt, 1993).

When CCP and CSP pressures are evaluated during erections, one can

distinguish a vascular-subsystolic tumescence phase that is followed by

development of suprasystolic pressure peaks. The subsystolic tumescence

phase is considered a purely vascular event characterized by passive filling of

the tissues with blood. In CSP pressure recordings this phase has been shown to

have the same pulsatile frequency as electrocardiogram recordings and is thus a

direct reflection of arterial blood flow (Schmidt, M. H. et al., 1995). The suprasystolic pressure peaks, however, are considered to be a result of muscular contractions that consequently augment penile rigidity. Pressure peaks that occur in the CCP are considered secondary to IC muscle contractions (Beckett et al.,

1973; Beckett et al., 1974; Purohit and Beckett, 1976) and pressure peaks in the

CSP have been shown to occur secondary to BS muscle contractions (Beckett et al., 1975; Schmidt, M. H. et al., 1995; Schmidt, M. H. et al., 1994). Muscular pressure peaks generated in the CCP reach pressures of 714 mmHg in rats

(Bernabe et al., 1999), 6530 mmHg in stallions (Beckett et al., 1973), 7434 mmHg in dogs (Purohit and Beckett, 1976), and 1100 mmHg in humans (Meehan and Goldstein, 1983). Muscular pressures generated in the CSP remain far below those of the CCP (approximate maximum of 600 – 700 mmHg), supporting the concept that during erections, pressures in the CCP are generated in a closed cavernous system, whereas the cavernous system of the CSP remains an open vascular system (Schmidt, M. H. and Schmidt, 1993). Since the pressures that are generated within these cavernous spaces are so large, it is essential that nonsaturating pressure transducer systems are used (Schmidt, M. H. and

Schmidt, 1993; Schmidt, M. H. et al., 1995).

Traditionally, monitoring of CCP pressure is considered the standard

method for evaluating erectile function since BS muscle activity and thus CSP

pressure is primarily associated with ejaculatory function. However, erections

appear to be consistently associated with pressure changes in the CSP

(Schmidt, M. H. et al., 1995; Schmidt, M. H. et al., 1994) suggesting that

monitoring of CSP pressure is also a valid method of assessing erectile events.

Moreover, monitoring CSP instead of CCP pressure carries two distinct

advantages. First, the bulb of the CSP is more vascular and contains less dense

fibrous tissue than the CCP allowing easier implantation and longer recording

times and second, preliminary data suggests that CSP recording allows for

simultaneous recording of erectile (Schmidt, M. H. et al., 1995) and micturition

events (Schmidt, M.H. et al., 2004).

Monitoring CCP and CSP pressure is currently performed using telemetry.

The technique of telemetry is based on transmission of AM radio waves that are

recorded, stored, and processed by software. More specifically, for recording

penile erections using the technique described by Giuliano et al. (1994) and

Schmidt et al. (1995) a pressure transducer is implanted in the CCP or CSP,

respectively and pressure changes are detected by the transducer and

transmitted to a receiver unit through AM radio waves. The receiver unit collects

these signals and transmits them to a computer system with software that

processes and stores the collected data (Figure 3.2).

Figure 3.2: Telemetry system diagram. This cartoon demonstrates the technique of telemetry. Radio waves from the implanted transducers are transmitted to a receiver unit and forwarded to a computer system with software that processes and stores the collected data. Courtesy of Jay Gizzi, Application Specialist, Data Sciences International, St. Paul, Minnesota.

The goal of this study was to verify that pressure waveforms obtained from the CSP by telemetry were consistently associated with either micturitions or penile erections. Furthermore we hypothesized that a positive relationship exists between the duration of the micturition pressure waveform and volume of urine expelled. Here we demonstrate the use of CSP pressure monitoring in the assessment of micturition and sexual function in 10 normal, freely-moving male rats.

3.2 Materials and Methods

Subjects

Ten adult, male Long-Evans hooded rats (Simonsen Laboratories, Gilroy,

CA, USA) age 70 ± 3 days (AVG ± SE), were used in this study. Rats were

housed individually in plastic cages, maintained on a 12 hour light/dark cycle,

and had free access to food and water. All animal experiments were conducted

after approval by the Institutional Laboratory Animal Care and Use Committee of

The Ohio State University and were performed in compliance with NIH guidelines

and recommendations.

Surgical Procedures and Post-Operative Care

Transducer implantation was carried out aseptically under deep

anesthesia induced by intraperitoneal administration of xylazine (TranquiVedTM,

Vedco Inc., St. Joseph, MO, USA; 10 mg/kg) and ketamine (ketamine HCl,

Abbott Laboratories, N.Chicago, IL, USA; 80 mg/kg). Anesthetic plane was

determined by withdrawal to foot pinch. A pre-operative dose of cefazolin (Ancef,

Abbott Laboratories, N.Chicago, IL, USA; 50 mg/kg) was administered

subcutaneously. Lacrilube ophthalmic ointment (Allergan Pharmaceuticals,

Irvine, CA, USA) was applied to the eyes prior to surgery and body temperature

was maintained at 37.5 ± 0.5ºC using a rectal thermal probe and heating pad.

The rat was placed in dorsal recumbency and surgical sites were shaved

and cleaned with betadine. A 3 cm skin incision was made on midline on the

raphe scroti and a 3 – 4 cm skin incision was made parallel to midline in the right

inguinal area. A telemetric pressure transducer catheter (TA11PA-C40, Data

Sciences International, St. Paul, MN, USA) as shown in Figure 3.3 was implanted

in the bulb of the CSP as described previously (Schmidt, M. H. et al., 1995).

Figure 3.3: TA11PA-C40 pressure transducer used in these studies.

Briefly, the battery of the transducer was placed subcutaneously in the lower right

abdomen and secured to the external oblique abdominal muscle with 2 sutures.

The catheter was tunneled subcutaneously to the perineal area. Testes were retracted and BS muscle and CSP were exposed (Figure 3.4).

Figure 3.4: Rat penile anatomy. This cartoon demonstrates proper placement of the transducer catheter tip within the bulb of the CSP. For illustration purposes the BS and IC muscles are removed on the right side of this figure but these muscles are left intact during the surgical procedure. BS = bulbospongiosus, CCP = corpus cavernosum of the penis, CSP = corpus spongiosum of the penis, IC = ischiocavernosus. Courtesy of Dr. Markus Schmidt.

With minimal retraction of the BS muscles, a guide hole was made into the

distal bulb of the CSP with a 21 gauge needle. The open tip of the catheter was

placed into the bulb of the CSP and secured with biological glue and 1 suture

(Figure 3.4). The scrotal incision was then closed in 1 layer and the inguinal

incision was closed in 2 layers. The rat was allowed to recover. Figure 3.5 demonstrates some aspects of this surgical procedure. Telemetric studies were

commenced 4 – 5 days post operatively.

A B C

Figure 3.5 A-C: Transducer implantation procedure. In A the scrubbed surgical area is shown. B shows the transducer battery in place and the catheter being held with forceps. In C the surgical area is shown approximately 14 days post operative.

CSP Pressure Recording

Physiological telemetry data were recorded on a PC computer using a

Spike2 data acquisition program (version 3.1, Cambridge Electronic Design

Limited, Science Park, Milton Road, Cambridge CB40FE, U.K.) (Figure 3.6 A).

Level of saturation of our system is 400 – 500 mmHg. The 10 animals underwent

2 x 24 hour CSP pressure recording while housed in a plastic cage with a wire mesh floor. Colored paper was mounted on a scale underneath the mesh floor of the cage and this was recorded on video. This made it possible to see when a micturition event occurred and the volume was based on change in scale readout

(1ml = 1mg). The receiver unit of the telemetry system was placed on top of the cage. This experimental condition is shown in Figure 3.6 B. To avoid time discrepancies, the camera time was recorded in a simultaneously running event channel of the acquisition program and used as the start time. In this way the 51

time and approximate volume of each micturition could be determined and

correlated to the pressure wave characteristics. During the dark hours a red light

was placed over the colored paper to facilitate detection of micturition events.

Four rats were recorded for a 24 hour period weekly for 2 months in order to determine quality of chronic recordings. These recordings were performed

while animals were housed in their regular cages as shown in Figure 3.6 A.

A B

Figure 3.6 A,B: Experimental condition for telemetry recording. In A the condition for recording CSP pressure in freely moving rats is shown. A cage is placed on top of a receiver and is connected to a computer system that collects and displays pressure waves. In B the condition is shown that was used to verify simultaneous occurrence of a micturition event on video and computer readout. As shown the receiver unit is placed on top of the cage allowing urine to fall on the scale.

CSP pressure recording during behavior tests was performed to verify

simultaneous occurrence of observed erectile events and wave forms

characteristic of erections on the computer readout. This was done by placing the

receiver unit underneath either the restraining cylinder during ex copulatory erection tests or underneath the cage in which mating tests were performed.

For all rats CSP pressure data were analyzed for the quantity, quality, and

time of occurrence of micturitions during the 24 hour period. During erection tests observed erectile events were recorded on the event channel during simultaneous data acquisition.

For 7 animals the duration of each micturition event was obtained from

CSP wave forms for 1 (2 rats) or 2 (5 rats) 24 hour time periods and was directly compared to the volume of that micturition obtained from the scale readout.

Behavioral Tests

Ex copulatory reflex erection tests:

Ex copulatory reflex erection tests were performed twice in 10 awake

animals as previously described (Hart, 1968; Holmes et al., 1988; Schmidt, M. H.

et al., 1995). Briefly, rats were placed on a board in dorsal recumbency with the

head and anterior torso in a loose-fitting restraining cylinder. The abdomen was

secured to the board with masking tape. This is shown in Figure 3.7 A. The head

and anterior torso could move freely in the cylinder. The telemetric receiver unit

was placed directly under the board. The preputial sheath was retracted, which is

the stimulus to elicit reflex erections, and maintained in retracted position by

placing the glans penis through a hole in a small piece of masking tape fastened

to the abdomen. Once the sheath was retracted, the reflex test lasted for 20

minutes. Rats were habituated to this procedure prior to surgery (2x10min and

2x20min) and the animals quickly adapt to the testing situation. Events were

visually scored according to previously described criteria (Schmidt, M. H. et al.,

1995): E1, weak glans engorgement; E2, moderate glans engorgement involving

some dilation of the distal glans; E3, intense flaring or cup of the distal glans

(Figure 3.7 B); F1, dorsiflexion of the penile body; F2, dorsiflexion or flip of the penile body greater than 90º with respect to the body of the rat. These reflexes were scored with a numerical keypad as events 1 – 5, respectively, and were recorded on the event channel during simultaneous data acquisition.

A B Figure 3.7 A,B: Ex copulatory reflex erection test. In A a rat is shown in the restraining cylinder. In B an erectile event (E3) is shown.

Non-contact erection tests:

Non-contact erection tests were performed twice in a glass aquarium (51 x

30 x 29), which was divided in half by a sheet of wire mesh. The male rat was

allowed 5 minutes to adjust to the new environment prior to the start of the test.

An estrous female rat was placed in one compartment and the male in the other.

The female rat had been administered estradiol cypionate (200μg = 0.1 ml SQ)

24 hours prior to the test and progesterone (500μg = 0.1 ml SQ) 6 hours before the test to ensure receptive behavior. Before the test, female receptivity was verified with non-experimental male rats. The rats were observed for 30 minutes.

Events (1: visible erections, 2: grooming of body parts, 3: grooming of genital area without visible erections) were recorded in the event channel as described above. This test was performed with the telemetric receiver unit placed directly under the aquarium.

Mating tests:

Mating tests were performed twice in a glass aquarium (51 x 30 x 29). The

male rat was allowed a 5 minute adjustment period prior to introduction of a

female rat in estrous. After the male and female rats were placed together they

were observed for 30 minutes. The female rat had received the same hormonal

treatment as described for the non contact erection test. Events (1: mounts, 2:

intromissions, 3: ejaculations, 4: grooming of body parts, 5: grooming of genital

area without visible erections) were recorded in the event channel as described

above.

This test was performed with the telemetric receiver unit placed directly

under the aquarium.

Statistics:

Data are presented as means ± standard error of the mean (SE). To compare the number of micturitions detected by telemetry with those detected by

video and to determine the relationship between duration and volume of

micturition, regression analysis was performed. Agreement between the two

methods was assessed by calculating bias (mean difference between the two

methods) and limits of agreement (2 x standard deviation = 2SD) (Bland, J.M.

and Altman, 1986). The Bland and Altman statistical method is the most

appropriate tool to compare agreement between repeated measurements by

each of 2 methods on the same subjects. Also, Bland and Altman plots are useful

to reveal a relationship between the differences and the averages, to look for

systematic bias, and to identify possible outliers (Bland, J.M. and Altman, 1986;

Bland, J. M. and Altman, 1999). Analysis of variance (ANOVA) was used to

identify significant differences in CSP waveform characteristics of erectile events

recorded during the 3 different behavioral contexts. The statistical computations

were performed with software packages (Sigmastat 3.0, SPSS, Chicago, IL and

SPSS 12.0, SPSS, Chicago, IL).

3.3 Results

Rats were 70 ± 3 days and weighed 300 ± 10 g for transducer

implantation. Complications that occurred around the transducer implantation site

were minor and included mild swelling, loss of hair, and scabbing, all of which

healed with time.

For all 10 rats micturition and erectile events were easily identified from

computer readouts and the characteristics of 3 types of events are described

below. Quality of chronic recordings remained stable in 3 of the 4 rats we

followed weekly for an 8 week period. Explanting of the transducer of the 4th rat revealed that the catheter tip was no longer in the CSP.

Micturition

Telemetrically obtained CSP pressure waveforms during a micturition event are characterized by a smooth, flaccid base line pressure on top of which a pulsatile pattern occurs with a highly conserved frequency of 9.1 ± 0.3 Hz and mean maximum pressure of 93.8 ± 8.9 mmHg. After-peaks (3.2 ± 0.3) are typically seen occurring every 3 – 5 seconds (Figure 3.8 A,B). In Figure 3.8B the phase during which urine is expelled and the afterpeaks are illustrated. During micturition events we do not recognize a vascular component in the CSP pressure waveforms; however, we recognize the muscular component of BS muscle contractions in the form of CSP pressure peaks. This is further demonstrated in Figure 3.9 which shows BS EMG recordings obtained during micturition events. Detailed characteristics of CSP pressures during micturition events are summarized in Table 3.1.

The number of micturitions seen on video and recorded by telemetry is shown for all rats for 2 x 24 hour periods (Figure 3.10). In Figure 3.11 the total number of micturitions (A) and the average number of micturitions per 24 hours

(B) that were seen on video and recorded by telemetry are shown.

31 Keyboard 200

190

180

170

160

150

140

130

120

110

100

R4 90 mmHg

20 21.0169

-10

-20 44414 44416 44418 44420 44422 44424 44426 44428 44430 44432 44434 44436 44438 44440 44442 444 s A

2 weeks post implantation

mm Hg “After” Peaks 80 Expulsion of urine

0 4 sec

Figure 3.8 A,B: CSP pressure waveform obtained by telemetry during micturition. In B the phase of expulsion of urine and the afterpeaks are indicated.

Figure 3.9 A-C: Electromyographic activity in the BS muscle during a micturition event. This figure shows BS muscle firing during the phase of urine expulsion. A – C are same tracings shown at different scales to clarify that BS muscle bursts occur at the same time as CSP pressure peaks. Data obtained by Dr. Markus Schmidt.

Event analysis Afterpeak analysis

Duration AUC Mean Max Number Peak Number Duration AUC Mean

P P Peaks Frequency P

3.6 ± 119 ± 33 ± 94 ± 32 ± 9.1 ± 0.3 3.2 ± 0.33 ± 15.3 46 ±

0.2 11.5 1.9 8.9 1.6 0.3 0.02 ± 2.4 2.2

Table 3.1: Micturition waveform characteristics in the adult Long-Evans rat. Analysis of 24 hour micturition events. Micturition waveform characteristics include duration (seconds), area under the curve (AUC)(mmHgxseconds), mean and maximum pressures (P)(mmHg), frequency (Hz) of the expulsion phase and number, duration (seconds), AUC (mmHgxseconds), and mean P of the afterpeaks.

Micturitions

90 80 70 60 50 Video 40 Telemetry 30 20 10 0 Micturition number / 24 h 24 / number Micturition

6 6 0 9 9 2 2 4 5 5 47 64 24 24 2 27 24 24 26 26 26 2 27 27 1 1 1 1247 1251 1251 1270 1 1 1 1 1 1 1 1269 1269 1274 1274 1 1 Rat number

Figure 3.10: Number of micturitions. Total number of micturitions per 24 hours shown for each rat detected by telemetry and by video observation.

Micturitions total number Micturitions per 24 h

700 40

600 35

30 500 25 400 Video Video 20 300 Telemetry Telemetry 15 200 10 Number (10 rats 48 h)48 rats Number (10 100 5

0 0 Number (AVG + SE) per rat per 24 h

A B

Figure 3.11 A,B: Total number of micturitions seen on video and detected by telemetry. In A the total number is shown and in B the mean + SE of micturitions per rat per 24 hours is given.

Linear regression analysis between telemetry and video observed number

of micturitions resulted in R = 0.977 (p<0.001; F=364). The bias was -1.9 and the

upper and lower limits of agreement were 7.5 and -11.3, respectively (Figure

3.12). One rat (1264) had 1 measurement outside the limits of agreement (-15).

Bland-Altman: Micturitions/24h

10 1.96xSD 5 0 Mean -5 0 20406080100

-10 -1.96xSD (number/24h) -15 Video - Telemetery Telemetery - Video -20 Mean (video + telemetry)/2 (number/24h)

Figure 3.12: Bland and Altman plot of the number of micturitions per 24 hours detected by video or telemetry. The average number of micturitions per 24 hours per rat (for 9 rats 2 x 24 h and for 1 rat 1 x 24 h) was used for this analysis.

The volume per micturition was 0.95 ± 0.12 ml/urination. For 7 rats we

determined the relationship between micturition pressure wave form duration and

volume of urine expelled. This is shown in Figure 3.13A. Linear regression analysis revealed R = 0.823 (p<0.001; F=545). The formula for the positive relationship between the two parameters is shown in Figure 3.13B after combination of all data.

Volume and Duration of Micturitions

3.5 1246 a 3 1246 b 2.5 1247 1251 a 2 1251 b 1.5 1249

Volume (ml) 1 1269 a 0.5 1269 b 0 1270 a 1270 b 0510151273 a Duration (sec) 1273 b

Volume and Duration of Micturitions

3.5 3 2.5 2 Combined data 1.5 Linear (Combined data)

Volume (ml) 1 y = 0.2754x - 0.0946 0.5 0 0 5 10 15 Duration (sec)

Figure 3.13 A,B: Relationship between duration and volume of micturition. The volume and duration of micturitions is shown for individual animals (A) and for the combined data (B).

Sexual Function

Telemetrically obtained CSP pressure waveforms during full erectile

events are characterized by development of a tumescence pressure of at least

30 mmHg above baseline on top of which 1 or many suprasystolic pressure peaks occur. At least 1 peak must reach a pressure of 100 mmHg above the tumescence pressure for the event to be classified as a full erectile event (Figure

3.14). The limit of 100 mmHg over baseline pressure is based on being

1.5xstandard deviation below the average maximum pressures during E1 events

(200 mmHg) (Dr. Markus Schmidt, personal communication). The limit of 100 mmHg over baseline pressure is at 1.5x standard deviation below 200 mmHg.

Events that reach pressures of 130 – 140 mmHg are thus considered partial erectile events. Partial erectile events are similar to full erectile events with the exception that peaks never reach 100 mmHg over the tumescence pressure

(Figure 3.15). These events clearly demonstrate the vascular subsystolic phase of erections due to cardiogenic vasodilatory mechanisms. In contrast, the full erectile events have characteristics of both the vascular subsystolic component and the muscular suprasystolic component.

During ex copulatory reflex erection tests 100% of observed erectile

events corresponded with CSP waveform patterns characteristic of erectile

events. Partial erectile events occurred infrequently and were associated with

partial filling of the base but not with erectile events.

31 Keyboard 300

280

260

240

220

200

180

160

R4 140 mmHg

120

100

20 21.0169

-20 45276 45278 45280 45282 45284 45286 45288 45290 45292 45294 45296 45298 45300 45302 45304 s

Figure 3.14: CSP pressure waveform obtained by telemetry during a full erectile event.

140

120

100

1 mmHg Pressure 60

20 20.0193

7274 7276 7278 7280 7282 7284 7286 7288 7290 7292 7294 7296 7298 7300 7302 7304 7306 7308 7310 s

Figure 3.15: CSP pressure waveform obtained by telemetry during a partial erectile event.

The characteristic muscular generated suprasystolic pressure peaks seen in the

CSP waveforms occurred during full erectile events with glans engorgement (E1

– E3). Figure 3.16 and Table 3.2 illustrate characteristics of CSP waveforms recorded during the different contexts studied.

400 Reflex E3 E3 E3 E3 300 E2 200 mmHg

100 E1

0 10 Sec 400

Non-contact 300

m 200

100

400 3 Sec

300 Intromission 200 mmHg

100

0 3 Sec

Figure 3.16: CSP pressure waveforms recorded during 3 behavioral contexts.

During non contact mating tests CSP pressure waveforms characteristic of full erectile events were observed during visible erectile events associated with grooming of the genital area. Furthermore, identically looking CSP pressure

waveforms were seen while the male rat was engaged in grooming or exploratory behavior, in spite of no visually confirmed observation of erections. During mating tests suprasystolic peaks were seen associated with various components of mating such as mounting, copulation, and intromission. However these erectile events were of significantly shorter duration (p<0.05) and therefore did not have the characteristic appearance of erectile events seen in other contexts such as

during grooming or ex copulatory reflex erection tests. Furthermore, during non contact and mating tests, more events were identified by telemetry than by

observation alone.

Context Duration AUC Mean P Max P Number Duration AUC Mean P

* ** Peaks * ** *

24 hour 25 ± 2.5 1401 ± 54 ± 1.1 320 ± 4.2 ± 0.73 ± 76 ± 3.6 108 ±

154 14 0.2 0.03 3.1

Reflex 54 ± 1.3 2855 ± 55 ± 3.1 333 ± 6.2 ± 0.82 ± 86 ± 10 106 ±

584 26 1.2 0.05 7.8

Non- 13 ± 2.3 622 ± 45 ± 5.1 235 ± 2.8 ± 0.50 ± 49 ± 8.1 94 ± 7.2

contact 165 41 0.9 0.09

Mating 4.2 ± 227 ± 56 ± 3.4 274 ± 1.7 ± 0.54 ± 55 ± 5.7 106 ±

1.1 54 22 0.5 0.05 5.2

Table 3.2: Erectile event waveform characteristics in the adult Long-Evans rat. Analysis of CSP pressure waveforms during erectile events. Waveform characteristics analyzed include duration (seconds), area under the curve (AUC)(mmHgxseconds), mean and maximum pressures (P)(mmHg), number of peaks of the event, and duration (seconds), AUC (mmHgxseconds), and mean P of the individual peaks. Significance levels: * p<0.05; ** p<0.01. 66

3.4 Discussion

In this study we demonstrate the accuracy of CSP pressure recording as a

modality for measuring frequency and quality of micturition and erectile events in

awake freely moving male rats. Furthermore duration of micturition appeared

predictive of micturition volume.

Transducer implantation is a minimally invasive surgery with no major

complications encountered during our study period of 2 months. Analysis of

pressure wave-forms that were recorded by telemetry is straightforward and the

3 types of events, micturition, full erectile and partial erectile events, are easily

distinguished and recorded. When pressures are analyzed and compared to other studies and/or compared between different behavioral contexts, it is important to determine the pressure at which the recording system saturates. We

do not believe saturation effects AUC since the period at which pressures could

be higher than the saturation pressure would be very small.

Based on BS EMG and CSP pressure recordings it appears that pressure

changes in the CSP during micturition events are due to muscle contractions of

the BS muscle. This represents a new finding, since traditionally the BS muscle

is only associated with ejaculation and its role in micturition has not been

investigated. Indeed, coordinated activation of the external urethral sphincter

(EUS) and contraction of the detrusor muscles are considered the main

processes involved in micturition (Pikov and Wrathall, 2001). We suggest that in

addition to the role the BS muscle plays in expelling seminal fluids, it likely also

plays a role in expelling urine. We propose that the BS muscle and the EUS

contract simultaneously. Thus, EUS muscle contractions may prevent backflow

of urine into the bladder while simultaneous BS muscle contractions assist in

expelling urine. Interestingly, the fact that BS muscle activity is important during

both micturition and erectile events is also demonstrated by the fact that, after

spinal cord injury in rats, it is not uncommon to see micturition events directly

followed by erectile events (Nout and Schmidt, unpublished observation). Reflex

patterns in the pelvic musculature are altered following spinal cord injury (Nout,

Y. S. et al., 2005a) and a consequence of this may be that BS muscle firing

during micturition enables the triggering of a subsequent reflex erectile event.

BS muscle contractions during micturition occur in a rapid pulsatile

manner and result in pressure peaks within the CSP characterized by a similar

pulsatile pattern. In contrast, the CSP pressure increase seen during partial

erectile events is consistent with a vascular pattern; the low amplitude pulsatile

pattern has a frequency that is directly associated with that of an

electrocardiogram (Schmidt, M. H. et al., 1995). The technique of CSP pressure determination directly measures this vascular component and forms an indirect measurement of the muscular component of the CSP pressure. CSP pressure changes during full erectile events reflect a combination of vascular subsystolic and muscular suprasystolic components. The vascular phase is characterized by development of a tumescence pressure that is increased from the baseline pressure. Suprasystolic pressure peaks occur on top of this tumescence pressure and are associated with BS muscle contractions. The fact that during full erectile events much higher pressures occur within the CSP than during

micturition is likely due to blood engorgement of the cavernous space during

erection whereas there is no filling of the CSP during micturition.

Simultaneous recording of CSP pressures and video-taping of animals

demonstrated an excellent agreement between the occurrences of micturition

events at the same time as the occurrence of a CSP pressure waveforms

characteristic for micturition. Slightly more micturitions were detected by

telemetry versus video observation resulting in an R of 0.98 instead of 1. We

speculate this is due to the fact that some micturition events were missed on

video either due to changing of the video tape while recording or due to poor

lighting during dark resulting in inability to see urine fall on the colored paper.

While animals were housed on wire mesh we found an average frequency

of 30 ± 4 micturition events per 24 hours. One other study that used metabolic

cages to determine frequency of micturition in male Sprague-Dawley rats found

they urinated approximately 21 times per 24 hours (Schmidt, F. et al., 2001).

However, while the rats in our study had not been accustomed to the study condition, the rats in Schmidt’s study had been acclimatized to the metabolic cages during a 7 day period prior to testing. When we examined micturition characteristics of male rats housed in their regular cages we found a micturition frequency of 26 ± 2 per 24 hours (Nout, Y. S. et al., 2005b), which is closer to what Schmidt et al (2001) reported. In this study we found a mean volume per micturition of 0.95 ± 0.12 ml. This is slightly less than what Schmidt et al (2001) reported. They found an average micturition volume of approximately 1.3 ml.

However, when combining these results, the total volume of urine expelled per

24 hours would be similar between the two studies. The rats in our study

apparently urinated smaller volumes more frequently, perhaps related to the

different housing environment or strain differences. Another study reported a

smaller volume (0.6 ml) per micturition in male Wistar rats examined in metabolic

cages (Haas et al., 1997). These rats has a smaller body weight (mean: 250 g)

compared to the rats we used (mean: 300 g) and the rats used by Schmidt et al

(2001) (mean: 399 g). It is likely that smaller rats produce a smaller volume per micturition. Since the number of micturition per 24 hours and the volume of micturitions in this study compared well to what has been found in rats that were observed in metabolic cages, we believe transducer implantation does not affect the micturition reflex.

Telemetric monitoring of erectile events appears a consistent and

accurate method for detecting these events in conscious single housed rats and

in awake rats during behavioral tests. From this study it was evident that erectile events are difficult to detect by eye in rats during non contact and particularly

during mating tests. Typically in behavioral testing conditions, we infer

intromission from a specific behavioral pattern although we observe neither

intromission nor erection. We suggest that the use of CSP pressure analysis is

more inclusive and less likely to miss important data. Critics may suggest we are

dealing with false positive erectile events; however, there was a 1 to 1

relationship between visually confirmed erections during ex copulatory reflex

erection tests and characteristic CSP pressure changes. Moreover, we never

observed erectile events without CSP pressure changes.

Significant differences were present in CSP pressure waveforms during

erectile events recorded in different behavioral contexts. This highlights the

importance of context-specificity of erectile events and suggests interpretation of

the various erectile events by telemetry should occur within the context of

specific experimental conditions. The differences in CSP pressures may be due

to differences in erectile physiology between the 3 behavioral contexts studied

here. Perhaps this reflects the fact that reflex erections are likely to be more

spinally mediated and erections that occur during intromission likely have more

supraspinal input. Further study of the characteristics of CSP waveforms during

erectile events in non contact and/or mating tests and their relationship with

observed behavior may improve our understanding of context specificity of

erectile events. Future research involving pressure monitoring in both CSP and

CCP is required to further elucidate the roles of both cavernous systems and of

the muscles controlling them during erectile events.

Telemetric monitoring of CSP pressure for assessment of micturition and

erectile events has clear benefits over traditional methods of examining these

functions in freely moving animals. CSP pressure monitoring allows simultaneous

assessment of micturition and erectile function, which is important for scientists

interested in more than 1 autonomic functional outcome. This method allows

examination of animals housed in a natural habitat and determination of events that are in general very hard to observe by eye or video recordings. Furthermore, recordings obtained by telemetry not only provide us with quantifiable data such as number of events per 24 hours, but also allows us to determine other

parameters of importance in assessment of these events, such as duration and pressures that occur during these events. Since the bulb of the CSP is more vascular than the CCP, implantation of transducers is easier and failure rate is less. Furthermore, ability to perform long-term measurements is an advantage of

CSP monitoring versus CCP monitoring.

In conclusion, changes of CSP pressure wave-form characteristics, detectable by telemetry, are a valuable tool for detecting micturition and erectile events in conscious freely moving rats. Micturition and erectile function are predominantly regulated by the autonomic nervous system and damage to these components following spinal cord injury results in alterations of these important physiologic functions. We anticipate the use of telemetric recording of CSP pressure will aid in future studies examining changes in autonomic outcome following SCI.

3.5 References

Anderson KD: Targeting recovery: priorities of the spinal cord-injured population. J Neurotrauma 21:1371-1383, 2004.

Beckett SD, Hudson RS, Walker DF, Reynolds TM, Vachon RI: Blood pressures and penile muscle activity in the stallion during coitus. Am J Physiol 225:1072-1075, 1973.

Beckett SD, Walker DF, Hudson RS, Reynolds TM, Purohit RC: Corpus spongiosum penis pressure and penile muscle activity in the stallion during coitus. Am J Vet Res 36:431-433, 1975.

Beckett SD, Walker DF, Hudson RS, Reynolds TM, Vachon RI: Corpus cavernosum penis pressure and penile muscle activity in the bull during coitus. Am J Vet Res 35:761-764, 1974.

Bernabe J, Rampin O, Sachs BD, Giuliano F: Intracavernous pressure during erection in rats: an integrative approach based on telemetric recording. Am J Physiol 276:R441-449, 1999.

Bland JM, Altman DG: Statistical methods for assessing agreement between two methods of clinical measurement. Lancet 1:307-310, 1986.

Bland JM, Altman DG: Measuring agreement in method comparison studies. Stat Methods Med Res 8:135-160, 1999.

Cannon TW, Damaser MS: Effects of anesthesia on cystometry and leak point pressure of the female rat. Life Sci 69:1193-1202, 2001.

Conte B, D'Aranno V, Santicioli P, Giuliani S, Mancinelli A, Furio M, Maggi CA, Meli A: New method for recording cystometrograms in conscious, freely moving rats. J Pharmacol Methods 19:57-61, 1988.

Giuliano F, Bernabe J, Rampin O, Courtois F, Benoit G, Rousseau JP: Telemetric monitoring of intracavernous pressure in freely moving rats during copulation. J Urol 152:1271-1274, 1994.

Haas M, Kluppel AC, Moolenaar F, Meijer DK, de Jong PE, de Zeeuw D: Urine collection in the freely moving rat: reliability for measurement of short-term renal effects. J Pharmacol Toxicol Methods 38:47-51, 1997.

Hart BL: Sexual reflexes and mating behavior in the male rat. J Comp Physiol Psychol 65:453- 460, 1968.

Holmes GM, Chapple WD, Leipheimer RE, Sachs BD: Electromyographic analysis of male rat perineal muscles during copulation and reflexive erections. Physiol Behav 49:1235-1246, 1991.

Holmes GM, Holmes DG, Sachs BD: An IBM-PC based data collection system for recording rodent sexual behavior and for general event recording. Physiol Behav 44:825-828, 1988.

Holmes GM, Sachs BD: Erectile function and bulbospongiosus EMG activity in estrogen- maintained castrated rats vary with behavioral context. Horm Behav 26:406-419, 1992.

Kruse MN, Belton AL, de Groat WC: Changes in bladder and external urethral sphincter function after spinal cord injury in the rat. Am J Physiol 264:R1157-1163, 1993.

Meehan JP, Goldstein AM: High pressure within corpus cavernosum in man during erection. Its probable mechanism. Urology 21:385-387, 1983.

Noreau L, Proulx P, Gagnon L, Drolet M, Laramee MT: Secondary impairments after spinal cord injury: a population-based study. Am J Phys Med Rehabil 79:526-535, 2000.

Nout YS, Schmidt MH, Tovar CA, Culp E, Beattie MS, Bresnahan JC: Telemetric monitoring of corpus spongiosum penis pressure in conscious rats for assessment of micturition and sexual function following spinal cord contusion injury. J Neurotrauma 22:429-441, 2005b.

Pikov V, Gillis RA, Jasmin L, Wrathall JR: Assessment of lower urinary tract functional deficit in rats with contusive spinal cord injury. J Neurotrauma 15:375-386, 1998.

Pikov V, Wrathall JR: Coordination of the bladder detrusor and the external urethral sphincter in a rat model of spinal cord injury: effect of injury severity. J Neurosci 21:559-569, 2001.

Purohit RC, Beckett SD: Penile pressures and muscle activity associated with erection and ejaculation in the dog. Am J Physiol 231:1343-1348, 1976. 73

Sachs BD: Penile responses of rats (Rattus norvegicus) in extended ex copula tests. J Comp Psychol 99:342-349, 1985.

Sachs BD, Akasofu K, Citron JH, Daniels SB, Natoli JH: Noncontact stimulation from estrous females evokes penile erection in rats. Physiol Behav 55:1073-1079, 1994.

Sakamoto K, Smith GM, Storer PD, Jones KJ, Damaser MS: Neuroregeneration and voiding behavior patterns after pudendal nerve crush in female rats. Neurourol Urodyn 19:311-321, 2000.

Schmidt F, Yoshimura Y, Ni RX, Kneesel S, Constantinou CE: Influence of gender on the diurnal variation of urine production and micturition characteristics of the rat. Neurourol Urodyn 20:287- 295, 2001.

Schmidt MH, Schmidt HS: The ischiocavernosus and bulbospongiosus muscles in mammalian penile rigidity. Sleep 16:171-183, 1993.

Schmidt MH, Valatx JL, Sakai K, Debilly G, Jouvet M: Corpus spongiosum penis pressure and perineal muscle activity during reflexive erections in the rat. Am J Physiol 269:R904-913, 1995.

Schmidt MH, Valatx JL, Schmidt HS, Wauquier A, Jouvet M: Experimental evidence of penile erections during paradoxical sleep in the rat. Neuroreport 5:561-564, 1994.

Yoshiyama M, Nezu FM, Yokoyama O, de Groat WC, Chancellor MB: Changes in micturition after spinal cord injury in conscious rats. Urology 54:929-933, 1999.

CHAPTER 4

TELEMETRIC MONITORING OF CORPUS SPONGIOSUM PENIS PRESSURE

IN CONSCIOUS RATS FOR ASSESSMENT OF MICTURITION AND SEXUAL

FUNCTION FOLLOWING SPINAL CORD CONTUSTION INJURY

(Published: J Neurotrauma 22 (4) 429 – 441)

4.1 Introduction

Spinal cord injury (SCI) results in disruption of motor, sensory, and autonomic function. In research models, recovery following SCI is generally assessed by examination of motor function, leaving autonomic function largely ignored. However, in humans, impairment of urogenital tract function following

SCI and its subsequent complications are highly prevalent and clinically very important (Anderson, 2004; Noreau et al., 2000). The state of bladder function and sexual reflexes is critical and continued investigation of pathophysiology and treatments to improve recovery of these autonomic functions are, therefore, essential.

Most studies that have examined micturition function following SCI in rat

models have been conducted using cystometric and electromyographic (EMG) 75

techniques in anesthetized animals or post-mortem examination of bladder

characteristics (Kruse et al., 1993; Pikov et al., 1998; Pikov and Wrathall, 2001).

However, both anesthesia and placement of catheters in bladder or urethra are known to alter the micturition reflex (Cheng et al., 1995; Yoshiyama et al., 1999).

Investigators of penile erection physiology have developed a technique in which pressures within the corpus cavernosum or corpus spongiosum of the penis can be monitored continuously in conscious animals (Giuliano et al., 1994; Schmidt,

M. H. et al., 1995; Schmidt, M. H. et al., 1994). Unlike in recordings from the corpus cavernosum, continuous monitoring of pressure in the bulb of the corpus spongiosum of the penis (CSP) in awake, freely moving rats allows for simultaneous recording of erectile (Schmidt, M. H. et al., 1995) and micturition events (Schmidt, M.H. et al., 2004). Monitoring of CSP pressure can be performed over extended periods of time (8 – 12 weeks) in conscious animals, and the procedures have recently been validated in normal rats (Schmidt, M.H. et al., 2004).

In the normal rat, micturition is regulated by spinal and supraspinal neural

pathways and is mediated through contraction of the bladder detrusor muscle

accompanied by coordinated activation of the external urethral sphincter (EUS)

(Kakizaki et al., 1997; Yoshiyama et al., 1999). Unlike in humans, micturition in normal rats is characterized by a pulsatile flow pattern that occurs through rapid, brief contractions and relaxations of the EUS, in addition to the simultaneous burst firing activity apparent in the bulbospongiosus (BS) muscles (Schmidt et al.,

2004). Following incomplete SCI, recovery of micturition occurs in rats and is 76

sufficiently effective that manual bladder expression is no longer required after a

variable time following SCI. However, this recovery is limited and voiding remains

inefficient with long-term changes occurring in muscle EMG patterns, micturition

frequency, volume, and bladder weight (Chancellor et al., 1994; Cheng et al.,

1999; Kruse et al., 1993; Yoshiyama et al., 2000; Yoshiyama et al., 1999).

Initially a period of spinal shock occurs during which the bladder is areflexive and overflow incontinence may be present. This is followed by the development of involuntary reflex micturition involving detrusor and EUS muscle activity, which are mediated by spinal reflex pathways (Cheng and de Groat, 2004; Kruse et al.,

1993). The returning detrusor muscle contractions may be hyperreflexic (Osborn et al., 1990) and inefficient voiding occurs if the EUS contracts simultaneously with the detrusor muscle, a condition referred to as detrusor-sphincter- dyssynergia (DSD). Although bursting activity of the EUS can be mediated by spinal mechanisms, it is less robust in rats with SCI than in rats with an intact neuraxis (Cheng and de Groat, 2004). Interestingly, the initial loss and subsequent recovery of reflex functions, which are autonomically mediated, show a similar pattern of recovery as locomotor function (Holmes et al., 2005) suggesting commonality of underlying recovery mechanisms.

The present study evaluates the use of CSP pressure monitoring as an

assessment tool for measuring recovery of autonomic nervous system function

after SCI. More specifically, we investigated the use of chronic telemetric

monitoring of CSP pressure in awake, freely moving rats to determine recovery

of both micturition and erectile events following a moderate spinal cord contusion

injury.

4.2 Materials and Methods

Subjects

Seven adult, male Long-Evans hooded rats (Simonsen Laboratories,

Gilroy, CA, USA) age 71 ± 2 days (AVG ± SE), weight 301 ± 7 g, were used in

this study. Rats were housed individually in plastic cages, maintained on a 12-h

light/dark cycle, and had free access to food and water. All animal experiments

were conducted after approval by the Institutional Laboratory Animal Care and

Use Committee of The Ohio State University and were performed in compliance

with NIH guidelines and recommendations.

Surgical Procedures and Post-Operative Care

Surgical procedures were carried out aseptically under deep anesthesia

induced by intraperitoneal administration of xylazine (TranquiVedTM, Vedco Inc.,

St. Joseph, MO, USA; 10 mg/kg) and ketamine (ketamine HCl, Abbott

Laboratories, N.Chicago, IL, USA; 80 mg/kg) for transducer implantation, and

pentobarbital (Abbott Laboratories, Chicago, IL, USA; 50 mg/kg) for SCI.

Anesthetic plane was determined by withdrawal to foot pinch. A pre-operative

dose of cefazolin (Ancef, Abbott Laboratories, N.Chicago, IL, USA; 50 mg/kg)

was administered subcutaneously. Lacrilube ophthalmic ointment (Allergan

Pharmaceuticals, Irvine, CA, USA) was applied to the eyes prior to surgery and

body temperature was maintained at 37.5 ± 0.5ºC using a rectal thermal probe and heating pad.

For transducer implantation the rat was placed in dorsal recumbency and surgical sites were shaved and cleaned with betadine. A 3 cm skin incision was made on midline on the raphe scroti and a 3 – 4 cm skin incision was made parallel to midline in the right inguinal area. A telemetric pressure transducer catheter (TA11PA-C40, Data Sciences International, St. Paul, MN, USA) was implanted in the bulb of the CSP as described previously (Schmidt, M. H. et al.,

1995). Briefly, the battery of the transducer was placed subcutaneously in the lower right abdomen and secured to the external oblique abdominal muscle with

2 sutures. The catheter was tunneled subcutaneously to the perineal area.

Testes were retracted and BS muscle and CSP were exposed. A guide hole was made into the CSP with a 21 gauge needle and the open tip of the catheter was placed into the bulb of the CSP and secured with biological glue and 1 suture.

The scrotal incision was then closed in 1 layer and the inguinal incision was closed in 2 layers. The rat was allowed to recover. Telemetric studies were commenced 4 – 5 days post-operatively.

A 12.5 g-cm spinal cord contusion injury (SCI) was delivered with a

MASCIS/NYU device as previously described (Gruner, 1992). Briefly, a dorsal laminectomy at T9 – T10 was performed and the spinal cord, with an intact dura mater, was impacted with a 10 g rod from a height of 12.5 mm. Post-operative care included administration of cefazolin subcutaneously for 7 days, twice daily bladder expression for 7 days and then once daily for 3 – 4 days, access to 1.2%

liquid vitamin C (Vedco, Inc., St. Joseph, MO, USA) prepared in sugar water, and fluid administration if animals had evidence of hematuria.

CSP Pressure Recording

Physiological telemetry data were recorded on a PC computer using a

Spike2 data acquisition program (version 3.1, Cambridge Electronic Design

Limited, Science Park, Milton Road, Cambridge CB40FE, U.K.). Animals underwent 2 x 24h CSP pressure baseline (BL) recording while housed in their cages prior to SCI. Following SCI, CSP pressures were recorded (24 h per day) on days 1-7, 10, 14, 17, 21, and 24. In addition to this, micturition volume was determined for a period 2 x 24 h prior to SCI and for 5 consecutive hours on days

3, 4, 5, 7, 10, 14 and 21 following SCI.

To determine micturition volume, rats were placed in a plastic cage with a mesh floor. Colored paper was mounted on a scale underneath the mesh floor of the cage and this was recorded on video. This made it possible to see when a micturition event occurred and the volume was based on change in scale readout

(1ml = 1mg). The receiver unit of the telemetry system was placed on top of the cage. To avoid time discrepancies, the camera time was recorded in a simultaneously running event channel of the acquisition program and used as the start time. In this way the time and approximate volume of each micturition could be determined and correlated to the pressure wave characteristics. Data were collected for 6 rats.

For 7 animals CSP pressure data were analyzed for the total number of

micturitions, full erectile events (tumescence pressure of at least 30 mmHg

above baseline on top of which at least 1 suprasystolic pressure peak of 100

mmHg above the tumescence pressure occurs), and partial erectile events

(similar to full erectile events with the exception that peaks never reach 100

mmHg over the tumescence pressure) in a 24-hour period. For 3 animals a

detailed waveform analysis was performed for 24 hour periods at BL and days 7

and 21 following SCI. Data analyzed were duration of the events, area under the curve (AUC), total number of pressure peaks, peak frequency and mean and maximum pressures. Furthermore, a peak analysis was performed. For micturition events this included the number of afterpeaks, and afterpeak duration,

AUC, and mean and maximum pressures. For erectile events this included the duration, AUC, and mean and maximum pressures of the peaks that occurred during those events.

Behavioral Tests

Ex copulatory reflex erection tests were performed in 6 awake animals as

previously described (Hart, 1968; Schmidt, M. H. et al., 1995). Briefly, rats were

placed on a board in dorsal recumbency with the head and anterior torso in a

loose-fitting restraining cylinder. The abdomen was secured to the board with

masking tape. The head and anterior torso could move freely in the cylinder. The

telemetric receiver unit was placed directly under the board. The preputial sheath

was retracted, which is the stimulus to elicit reflex erections, and maintained in

retracted position by placing the glans penis through a hole in a small piece of masking tape fastened to the abdomen. Once the sheath was retracted, the reflex test lasted for 20 min. Rats were habituated to this procedure prior to surgery and the animals quickly adapt to the testing situation. Events were visually scored according to previously described criteria (Schmidt, M. H. et al.,

1995): E1, weak glans engorgement; E2, moderate glans engorgement involving some dilation of the distal glans; E3, intense flaring or cup of the distal glans; F1, dorsiflexion of the penile body; F2, dorsiflexion or flip of the penile body greater than 90º with respect to the body of the rat. These reflexes were scored with a numerical keypad as events 1 – 5, respectively, and were recorded on the event channel during simultaneous data acquisition. Reflex testing was performed twice prior to SCI and on days 1, 3, 7, 14, and 21 following SCI.

Open-field walking was evaluated before and after transducer implantation, and at 48 hours, 5, 8, 16, and 22 days following SCI using the 21- point BBB Locomotor Rating Scale (Basso et al., 1995).

Histology

On day 25 after SCI, rats were deeply anesthetized with ketamine/xylazine followed by transcardiac perfusion with 0.9% NaCl and 4% formalin. The lesion center of the spinal cord was isolated and kept in formalin (4%) for 24 hours and then in sucrose (30%) for 24 – 48 hours. The tissue was frozen at –80ºC until further analysis. Lesion centers were sectioned transversely on a cryostat and stained with luxol fast blue for myelin and cresyl violet to reveal Nissle substance.

Sparing of the white matter in the lesion center was determined with computer

software (MCIDTM, Imaging Research Inc., St. Catherines, Ontario, Canada).

Statistics

Data are presented as means ± standard error of the mean (SE). A one-

way repeated measures analysis of variance (ANOVA) was used to analyze the

CSP pressure data. The null hypothesis was rejected at α = 0.05. Significant

differences identified by the ANOVA were isolated using the Student-Newman-

Keuls method for pairwise multiple comparison post-hoc test. To better

characterize the waveform changes over time following SCI, we performed trend

analyses on the detailed waveform data. Locomotor recovery data were analyzed

with the Friedman repeated measures ANOVA on Ranks followed by Dunn’s

method post-hoc test. The statistical computations were performed with software

packages (Sigmastat 3.0, SPSS, Chicago, IL and SPSS 12.0, SPSS, Chicago,

IL).

4.3 Results

Rats were 71 ± 2 days and weighed 301 ± 7 g for transducer implantation.

Rats were 82 ± 2 days and weighed 333 ± 5 g at the time of SCI. Complications

that occurred around the transducer implantation site were minor and included occasional mild swelling, loss of hair, and scabbing. One animal was sacrificed on day 5 after SCI due to bladder rupture secondary to manual bladder expression.

Micturition

Baseline data resulted in an average of 26 ± 2 micturition events per 24 hours per rat. The average volume of urine expelled per micturition was 1.0 ± 0.2 ml. CSP pressure waveforms during a micturition event were characterized by a smooth baseline pressure on top of which a pulsatile pattern occurred with a frequency of 9.0 ± 0.2 Hz. This waveform was generally followed by 3 – 4 afterpeaks (Figure 4.1). Following SCI bladders were manually expressed twice daily for 7 days and then once daily for 3 – 4 days. First spontaneous micturition events following SCI were seen by telemetry on days 2 (Rats 4 and 5), 3 (Rats 1 and 3), 4 (Rat 6), and 5 (Rat 2, 7). First micturition events detected following SCI were recognized by the characteristic pulsatile pattern, however, when compared to BL, the amplitude was low and afterpeaks were absent (Figure 4.2). A gradual return of the micturition motor pattern was seen, and 24 – 48 hours after onset of micturition events, afterpeaks were present again (Figure 4.2). Recovery of the total number of micturition events is shown in Figure 4.3. Total number of micturition events on day 24 following SCI was 19 ± 3.

Figure 4.1: Representative waveforms of erectile and micturition events. Waveforms characteristic of full erectile and micturition events at baseline and on day 7 and day 21 following SCI. CSP pressure in mmHg (y-Axis) over a 25 second interval (x-Axis). (Bars = 2.9s for erections and 3.8s for micturitions) 85

Figure 4.2: Representative micturition waveforms following SCI. Waveforms characteristic of return of micturition over the first 24 – 48 hours after the onset of the first micturition following SCI. Initially (acute recovery) a low amplitude pulsatile pattern is seen, after which (subacute recovery) an increased amplitude of the pulsatile pattern occurs. Finally, afterpeaks return following the pulsatile waveform, to complete the recovery of the micturition motor pattern. CSP pressure in mmHg (y-Axis) given for a 24 second interval (x-Axis).

Recovery of micturition

) 25 20 15 10

Number (24 h Number (24 5 0 BL12345671014172124 Day

Figure 4.3: Total number of micturitions following SCI. Total number of micturitions per 24-hour period at baseline (BL) and up to 24 days following SCI (n = 7 until day 6, then n = 6). *: significantly different from BL and days 4 – 24.

Video recording of rats on days 3, 4 and 5 following SCI revealed overflow

incontinence present in 5 of 6 rats on day 3 and in 2 rats on day 4. Overflow incontinence, characterized by continuous dribbling of urine in combination with a

(partial) full bladder, was not detectable by telemetry. All micturition events that

were seen on video corresponded with the CSP pressure wave characteristic of

micturition, detectable by telemetry. Urine volumes (ml) following SCI were

smaller than before SCI on days 3 (0.0 ± 0.0), 4 (0.5 ± 0.3), and 5 (0.8 ± 0.3)

before they gradually increased to reach an average volume of 2.1 ± 0.1 on day

21, which was significantly larger than before SCI (Figure 4.4).

Volume per micturition

2.5 ?

) 2 1.5 1

Volume (ml 0.5 0 BL 3 4 5 7 10 14 21 Day

Figure 4.4: Urine volume per micturition. Urine volume per micturition (ml) at baseline (BL) and up to 21 days following SCI (n = 6 until day 6, then n = 5). *: significantly different from BL; ▲: significantly different from day 3; ♦: significantly different from day 4; ■: significantly different from day 5.

Although the micturition CSP pressure waveforms following SCI remained similar to those seen before SCI, some differences were noted on days 7 and 21

(Figure 4.1). Table 4.1 shows detailed waveform characteristics for BL and days

7 and 21 following SCI. Significant linear trends were demonstrated towards increased micturition duration, AUC, mean pressure, number of peaks, and peak frequency over time (Fs > 18.5, p < 0.04). Although there were no significant changes in afterpeak analysis on days 7and 21, there was a transient absence of afterpeaks during recovery.

Day Duration AUC Mean Number Peak Number of Afterpeak

(s) * (mmHg x Pressure of Frequency Afterpeaks Duration (s)

s) * (mmHg) * Peaks * (Hz) *

BL 3.6 ± 0.3 127 ± 35 ± 1.9 32 ± 2.0 9.0 ± 0.2 3.4 ± 0.3 0.32 ± 0.03

12.5

7 6.1 ± 0.3 263 ± 43 ± 2.0 57 ± 1.6 9.9 ± 0.3 3.7 ± 1.0 0.26 ± 0.03

25.5

21 6.5 ± 0.7 302 ± 46 ± 1.6 66 ± 6.4 10.2 ± 0.1 3.9 ± 0.4 0.25 ± 0.01

37.1

Table 4.1: Micturition waveform characteristics. Micturition waveform characteristics (means ± SE) at baseline (BL) and on days 7 and 21 following SCI (n = 3). * : significant linear trends.

Erectile Events

The number of total erectile events with the proportion of full and partial erectile events is shown in Figure 4.5. During full erectile events a tumescence pressure develops of at least 30 mmHg above baseline on top of which 1 or many suprasystolic pressure peaks occur. At least 1 peak must reach a pressure of 100 mmHg above the tumescence pressure for the event to be classified as a full erectile event (Figure 4.1). Before SCI the number of full erectile events was

53 ± 4 per 24 hours. Following SCI the number of full erectile events was significantly less than BL for all time points, except for on day 2 (Figure 4.5).

Although the CSP pressure waveforms characteristic for full erectile events seen following SCI remained similar to those observed before SCI, some changes were seen on days 7 and 21 (Figure 4.1). 89

Erectile events Total erectile events Full erectile events Partial erectile events 80 70 60 r 50 40 30 Numbe 20 10 0 BL12345671014172124 Day

Figure 4.5: Total number of erectile events following SCI. Total number of erectile events per 24-hour period given with proportions of full and partial erectile events at baseline (BL) and up to 24 days following SCI (n = 7 until day 6, then n = 6). *: significantly different from BL and day 2. ▲: significantly different from days 1 – 24.

Table 4.2 shows detailed waveform characteristics for BL and days 7 and

21 following SCI. Significant linear trends were demonstrated towards increased

AUC (F = 4655, p < 0.00) and maximum pressure (F = 30.3; p < 0.03) of the full erectile events over time, and towards increased duration (Fs = 42.92, p < 0.03),

AUC (Fs = 53.56, p < 0.02), and average maximum pressure (Fs = 36.95, p <

0.03) of the individual suprasystolic pressure peaks over time. Furthermore, a

marginally significant linear trend was seen towards an increase in duration of full

erectile events (Fs = 17.76, p = 0.052).

Day Duration AUC Mean Maximum Peak Peak AUC Peak

(s) (mmHg x Pressure Pressure Duration (mmHg x s) Maximum

s) * (mmHg) (mm Hg) (s) * * Pressure

* (mmHg) *

BL 21.5 ± 1.1 1160 ± 87 53 ± 1.4 302 ± 20 0.68 ± 0.02 70 ± 1.2 252.3 ±

7 25.6 ± 3.6 1540 ± 59 ± 1.7 333 ± 34 0.74 ± 0.04 87 ± 4.6 275.6 ±

215 29

21 26.2 ± 1.8 1495 ± 84 57 ± 1.1 352 ± 11 0.93 ± 0.06 105 ± 3.6 289.9 ±

Table 4.2: Characteristics of full erectile event waveforms. Full erectile event waveform characteristics (means ± SE) at baseline (BL) and on days 7 and 21 following SCI (n = 3). * : significant linear trends.

Partial erectile events are similar to full erectile events with the exception that peaks never reach 100 mmHg over the tumescence pressure. The number of partial erectile events prior to SCI was 8 ± 2 per 24 hours. Following SCI this decreased and the total number of partial erectile events remained significantly less than at BL throughout the study period (Figure 4.5).

Reflex Erection Tests

Following SCI, latency (time of first erectile event following sheath

retraction) significantly decreased at all time points (Figure 4.6 A). The number of

erectile events that occurred during the testing period increased significantly on

days 7 and 21 following SCI. Moreover, the number of suprasystolic pressure 91

peaks that occurred within such events, increased significantly from BL on days

1, 3, 7, and 21 following SCI (Figure 4.6 B,C).

Latency Number of pressure peaks and erectile events

1200 60 1000 r 800 40 600 20 Numbe

Time (s) Time 400 200 0 0 BL 1 3 7 14 21 BL 1 3 7 14 21 Day Day Pressure peaks Erectile events A B

Pressure peaks per event

r 6 4

Numbe 2 0

BL 1 3 7 14 21 Day

Figure 4.6 A-C: Reflex erection test results. Reflex erection test results at baseline (BL) and up to day 21 following SCI (n = 6 until day 6, then n = 5). Latency, or time to first erectile event (A). Total number of pressure peaks and total number of erectile events (B). Number of pressure peaks per erectile events (C). *: significantly different from BL. 92

Locomotor Function

The 12.5 g-cm SCI resulted in an initial profound paraplegia in all 7 rats

characterized by a mean BBB locomotor score of 2.6 ± 0.7 at 48 hours after

injury. Gradually, rats showed recovery of locomotor function with frequent to

consistent weight-supported stepping and no forelimb-hindlimb coordination by

22 days following SCI. A mean BBB score of 10.7 ± 0.3 was reached at that time

(Figure 4.7).

Locomotor recovery

25 20 15 10

BBB - score BBB - 5 0 BL 2 5 8 15 22 Day

Figure 4.7: Recovery of locomotion function. Recovery of locomotor function according to the BBB Locomotor Rating Scale (n = 7 until day 6, then n = 6). *: significantly different from BL; ▲: significantly different from 48 hours.

Histopathology

No residual gray matter was observed at the lesion center, but a thin rim

of white matter remained. White matter sparing was determined in transverse sections of the spinal cord at the level of the lesion center, and occupied 19.4 ±

2.5% of the cross sectional area (Figure 4.8). 93

Figure 4.8: Spared tissue at lesion epicenter. Spared tissue at lesion epicenter shown for 6 animals, assessed by examination of transverse sections stained with luxol fast blue and cresyl violet.

4.4 Discussion

In this study we demonstrate that CSP pressure recording is a valuable

method for assessment of autonomic nervous system function following SCI.

Moreover, this technique allows determination of many parameters of micturition

and erectile events in conscious animals and enables us to distinguish subtle

differences within these events. Transducer implantation is a minimally invasive

surgery with no major complications encountered during our study period of 24

days following SCI. Analysis of pressure waveforms is straightforward and the 3

types of events (micturition, full erectile and partial erectile events) are easily

distinguished, since they occur on top of a baseline pressure that barely 94

fluctuates and generally lies between 0 – 10mmHg. The detailed waveform analysis is more complex; however, further development of the analysis software

may facilitate this in the future. This technique has been previously validated for

use in detection of erectile events (Schmidt, M. H. et al., 1995) and micturitions

(Schmidt, M.H. et al., 2004), and the present data further validate the use of this

technique in rats following SCI through simultaneous recording of CSP pressures

and both video recording of micturition events and performance of reflex erection

tests ((Nout et al., 2004).

The moderate SCI that rats were subjected to in this study resulted in

locomotor and histopathologic changes consistent with what has been shown

previously for this level of injury (Basso et al., 1995, 1996). Continuous dribbling of urine was seen on video recordings within the first 3 – 4 days following SCI, which is similar to what has been shown in previous studies (Chancellor et al.,

1994). As expected, overflow incontinence did not result in changes of CSP pressures and was thus not detectable by telemetry. However, a return of the autonomic/somatic motor pattern characteristic of the pre-lesion pulsatile flow of urine was easily identifiable using the telemetric device by monitoring pressures within the bulb of the CSP. Although first micturition waveforms were seen on days 2 – 5, normalization of the micturition frequency and return of the afterpeaks, only occurred around days 5 – 6, suggesting that twice daily post- operative manual bladder expression for a period of at least 6 days is necessary for this severity level of SCI. The total number of micturitions over a 24-hour period remained lower than at BL; however, this was not statistically significant.

The most significant findings related to micturition in the present

experiment were a 2-fold increase in volume per micturition at 10 days following

SCI and a 2-fold increased duration of micturition. Furthermore, we found

significantly increased mean pressures during micturition events at 7 and 21

days following SCI. These results are similar to what has been shown by other

investigators. Yoshiyama et al. (1999) demonstrated a strong correlation

between duration of bladder wall contraction and voided volume, both of which

were significantly increased following SCI. Furthermore, they showed a 4-fold

increase in volume threshold to induce micturition, determined by

cystometrograms, at 14 – 21 days following spinal cord transection. Chancellor et al. (1994) showed an almost 2-fold increase in urine volume per micturition at 1 month following a 50 g-cm SCI and found an increase in total voided volume per

24 hours without significant change in water intake. In addition, Mitsui et al.

(2003) demonstrated an increase in volume per micturition at 14 days post injury, however this difference did not become significant until 28 days post injury. In contrast, a study that utilized cystometrograms for assessment of urodynamics demonstrated a decrease in voided volume at 12 – 14 days following a 25 g-cm

SCI, although by that time the total bladder volume based on post-mortem analyses was increased 8-fold (Pikov et al., 1998).

A significant decrease in voiding efficiency resulting in increased residual

urine and increased bladder capacity is commonly seen in rats following SCI.

Multiple investigators have shown increased bladder weights, bladder wall

muscle hypertrophy, and increased voiding pressures associated with this

decrease in voiding efficiency (Pikov et al., 1998; Pikov and Wrathall, 2001;

Yoshiyama et al., 1999). Bladder weights may increase approximately 4-fold by day 12 – 14 following a mild SCI (Pikov et al., 1998).

Detrusor-sphincter-dyssynergia (DSD) is thought to be the main reason for

the observed increased voiding pressures and decreased voiding efficiency, and

has been documented in rats (Cheng and de Groat, 2004; Kruse et al., 1993;

Pikov et al., 1998; Yoshiyama et al., 2000). The degree of chronic impairment of detrusor-EUS coordination is positively correlated with the severity of the SCI

(Pikov and Wrathall, 2001). Tonic EUS activity, reflecting closure of the urethral outlet could be considered equivalent to DSD in rats, whereas EUS bursting activity, reflecting opening and closure of the urethral outlet reflects normal coordinated detrusor-sphincter function. Although EUS bursting activity can be mediated by spinal reflex mechanisms, the bursting activity that is seen after SCI is abnormal, consisting of shorter urethral opening times and this presumably contributes to the inefficient voiding and increased voiding pressures (Cheng and de Groat, 2004). Also, the amplitude from EUS EMG recordings has been shown to increase following SCI (Pikov and Wrathall, 2001). Suppression of EUS activity results in improvement of all voiding parameters (Kruse et al., 1993; Yoshiyama et al., 2000).

In addition to DSD, detrusor hyperreflexia has been well characterized in

rats following SCI (Mitsui et al., 2003) and is thought to be due to a lack of

supraspinal inhibition with or without an increase of afferent signaling. Moreover, enlargement of the bladder may result in plasticity of afferents, further

contributing to this hyperreflexia. The significance of this syndrome in the

recovery of micturition, however, is unknown. Recovery of micturition function

has been observed to occur through amelioration of DSD without noticeable

change of detrusor hyperreflexia (Mitsui et al., 2003). Also, it has been shown

that the intercontraction interval of the detrusor muscle may be reduced following

SCI (Pikov et al., 1998). When using cystometrograms, reduction of the pressure

threshold for micturition (Yoshiyama et al., 1999) and subsequently a quicker onset of micturition after filling of the bladder (Mitsui et al., 2003) is seen,

potentially as a result of detrusor hyperreflexia. However, volume threshold was

significantly increased and intravesicular pressures during voiding were 85%

higher in rats following SCI which, accompanied by reduced urine flow rates,

resulted in a significant decrease of voiding efficiency (Yoshiyama et al., 1999).

Hyperreflexia of the external anal sphincter (EAS) reflex has been shown

to occur following SCI (Holmes et al., 1998), in addition to DSD and detrusor

hyperreflexia, and is thought to be the main contributor to the defecatory

dysfunction that is seen following SCI. We suggest that in addition to these

muscles, other pelvic floor muscles, and specifically the BS musculature may

develop hyperreflexia or spasticity following SCI. This change in reflex activity is

reminiscent of hyperreflexia observed in other segmentally mediated reflexes

(Bose et al., 2002). In the current study we found trends towards increased mean

pressures, increased duration, increased AUC, and increased peak frequencies

of micturition events following SCI. These trends of increased CSP pressures

may serve as objective indicators of BS muscle spasticity, since pressure within

the bulb of the CSP is directly dependent on the surrounding BS musculature.

The telemetric technique employed in the present study allows long-term follow

up of these parameters in conscious rats.

The most significant changes related to erectile events observed in this

study were a decreased number of both full and partial erectile events per 24-

hour period following SCI, and a shortened latency to induction of erectile events

during reflex erection tests. Furthermore we found significant trends towards increased duration, AUC, and maximum pressures of the events as well as

trends towards increased peak duration, peak AUC, and peak maximum

pressures, which may result from BS muscle spasticity or hyperreflexia and may

prove a useful outcome measurement in future studies in addition to waveform

changes of micturition events. Normal, uninjured Long-Evans rats typically

display a long erection latency, as seen in our study (Holmes et al., 2001). The

significant reduction of erection latency that we observed following SCI is a result

of decreased supraspinal inhibitory input and is consistent with other reports

(Hart, 1968; Holmes et al., 2001; Hubscher and Johnson, 2000). Interestingly,

the total number of full and partial erections per 24-hour period detected by

telemetry decreased following SCI. We hypothesize that the number of partial

erectile events decreased due to disinhibition of supraspinal input allowing

almost all initiated erectile events to develop into full erections. We had expected

to find a large variation in the total number of erections following SCI secondary

to disinhibition of supraspinal input combined with dragging of the abdomen and

genital area following SCI. It has been shown that movement following SCI

affects the number of erectile events recorded in rats. Rats that move more and

consequently drag their abdomen more, have more erectile events than rats that

move little (Schmidt, M. H. et al., 1999). Little stimulation of the genital area is necessary to elicit erectile responses following SCI (Hart, 1968), as also demonstrated by the decreased latency in reflex erection tests. We suspect that the decreased number of erectile events on day 1 following SCI was due to generalized post-operative immobility.

Penile erection is only a single component of the complex male sexual

behavior and, in animals, can occur in response to tactile, visual, olfactory, and

auditory stimuli. In rats, erectile events have been demonstrated to occur during

copulatory tests (Hart, 1968), exposure to an estrous female (Sachs et al., 1994), ex copulatory reflex tests (Hart, 1968; Sachs, 1985), and paradoxical sleep

(Schmidt, M. H. et al., 1999; Schmidt, M. H. et al., 1994). Although grooming is commonly seen associated with erectile activity, grooming tends to follow the onset of erections, and has not been shown to elicit erectile events in freely moving rats (Sachs et al., 1988). However, one of the possible explanations of our finding of reduced erectile events following SCI is the assumption that erections occur secondary to genital grooming, which reduces markedly following

SCI. This could then result in a reduction of erectile events following SCI. Also, ascending sensory pathways from the male genitalia, which are bilaterally located within the dorsal quadrant at the midthoracic level of the spinal cord, are damaged in this model of SCI (Hubscher and Johnson, 1999). The other explanation for a reduction of erectile events following SCI would be through

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disruption of descending excitatory mechanisms in addition to disruption of inhibitory tracts. Although, most studies have concluded the descending tracts that modulate sexual reflexes to be inhibitory in nature, recent studies have demonstrated the significance of descending excitatory tracts particularly from the paraventricular nucleus of the hypothalamus (Giuliano and Rampin, 2000).

In conclusion, the current study shows the utility of chronic CSP pressure monitoring for evaluation of recording of both micturition and erectile function after SCI. The measures described reflect coordinated functioning of both somatic and autonomic components of the nervous system, and have been little studied in models of SCI. These functions are important therapeutic targets as they represent critical problems for the spinal cord injured population (Anderson,

2004). This technique may provide valuable objective outcome measurement data in future studies.

4.5 References

Anderson KD: Targeting recovery: priorities of the spinal cord-injured population. J Neurotrauma 21:1371-1383, 2004.

Basso DM, Beattie MS, Bresnahan JC: A sensitive and reliable locomotor rating scale for open field testing in rats. J Neurotrauma 12:1-21, 1995.

Basso DM, Beattie MS, Bresnahan JC: Graded histological and locomotor outcomes after spinal cord contusion using the NYU weight-drop device versus transection. Exp Neurol 139:244-256, 1996.

Bose P, Parmer R, Thompson FJ: Velocity-dependent ankle torque in rats after contusion injury of the midthoracic spinal cord: time course. J Neurotrauma 19:1231-1249, 2002.

Chancellor MB, Rivas DA, Huang B, Kelly G, Salzman SK: Micturition patterns after spinal trauma as a measure of autonomic functional recovery. J Urol 151:250-254, 1994.

Cheng CL, de Groat WC: The role of capsaicin-sensitive afferent fibers in the lower urinary tract dysfunction induced by chronic spinal cord injury in rats. Exp Neurol 187:445-454, 2004.

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Cheng CL, Liu JC, Chang SY, Ma CP, de Groat WC: Effect of capsaicin on the micturition reflex in normal and chronic spinal cord-injured cats. Am J Physiol 277:R786-794, 1999.

Cheng CL, Ma CP, de Groat WC: Effect of capsaicin on micturition and associated reflexes in chronic spinal rats. Brain Res 678:40-48, 1995.

Giuliano F, Bernabe J, Rampin O, Courtois F, Benoit G, Rousseau JP: Telemetric monitoring of intracavernous pressure in freely moving rats during copulation. J Urol 152:1271-1274, 1994.

Giuliano F, Rampin O: Central neural regulation of penile erection. Neurosci Biobehav Rev 24:517-533, 2000.

Gruner JA: A monitored contusion model of spinal cord injury in the rat. J Neurotrauma 9:123- 126; discussion 126-128, 1992.

Hart BL: Sexual reflexes and mating behavior in the male rat. J Comp Physiol Psychol 65:453- 460, 1968.

Holmes GM, Bresnahan JC, Beattie MS: Inhibition of pudendal reflexes in spinal rats. Reassessing the role of serotonin. Physiol Behav 74:57-64, 2001.

Holmes GM, Rogers RC, Bresnahan JC, Beattie MS: External anal sphincter hyperreflexia following spinal transection in the rat. J Neurotrauma 15:451-457, 1998.

Holmes GM, Van Meter MJ, Beattie MS, Bresnahan JC: Serotonergic fiber sprouting to external anal sphincter motoneurons after spinal cord contusion. Exp Neurol 193:29-42, 2005.

Hubscher CH, Johnson RD: Effects of acute and chronic midthoracic spinal cord injury on neural circuits for male sexual function. I. Ascending pathways. J Neurophysiol 82:1381-1389, 1999.

Hubscher CH, Johnson RD: Effects of acute and chronic midthoracic spinal cord injury on neural circuits for male sexual function. II. Descending pathways. J Neurophysiol 83:2508-2518, 2000.

Kakizaki H, Fraser MO, De Groat WC: Reflex pathways controlling urethral striated and smooth muscle function in the male rat. Am J Physiol 272:R1647-1656, 1997.

Kruse MN, Belton AL, de Groat WC: Changes in bladder and external urethral sphincter function after spinal cord injury in the rat. Am J Physiol 264:R1157-1163, 1993.

Noreau L, Proulx P, Gagnon L, Drolet M, Laramee MT: Secondary impairments after spinal cord injury: a population-based study. Am J Phys Med Rehabil 79:526-535, 2000.

Osborn JW, Taylor RF, Schramm LP: Chronic cervical spinal cord injury and autonomic hyperreflexia in rats. Am J Physiol 258:R169-174, 1990.

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Pikov V, Gillis RA, Jasmin L, Wrathall JR: Assessment of lower urinary tract functional deficit in rats with contusive spinal cord injury. J Neurotrauma 15:375-386, 1998.

Pikov V, Wrathall JR: Coordination of the bladder detrusor and the external urethral sphincter in a rat model of spinal cord injury: effect of injury severity. J Neurosci 21:559-569, 2001.

Sachs BD: Penile responses of rats (Rattus norvegicus) in extended ex copula tests. J Comp Psychol 99:342-349, 1985.

Sachs BD, Akasofu K, Citron JH, Daniels SB, Natoli JH: Noncontact stimulation from estrous females evokes penile erection in rats. Physiol Behav 55:1073-1079, 1994.

Sachs BD, Clark JT, Molloy AG, Bitran D, Holmes GM: Relation of autogrooming to sexual behavior in male rats. Physiol Behav 43:637-643, 1988.

Schmidt MH, Sakai K, Valatx JL, Jouvet M: The effects of spinal or mesencephalic transections on sleep-related erections and ex-copula penile reflexes in the rat. Sleep 22:409-418, 1999.

Schmidt MH, Valatx JL, Sakai K, Debilly G, Jouvet M: Corpus spongiosum penis pressure and perineal muscle activity during reflexive erections in the rat. Am J Physiol 269:R904-913, 1995.

Schmidt MH, Valatx JL, Schmidt HS, Wauquier A, Jouvet M: Experimental evidence of penile erections during paradoxical sleep in the rat. Neuroreport 5:561-564, 1994.

Yoshiyama M, Nezu FM, Yokoyama O, de Groat WC, Chancellor MB: Changes in micturition after spinal cord injury in conscious rats. Urology 54:929-933, 1999.

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CHAPTER 5

EFFECTS OF TRANSPLANTING GLIAL RESTRICTED PRECURSOR CELLS

AND ELEVATING cAMP CONCENTRATIONS ON AUTONOMICALLY

REGULATED BEHAVIOR AND HISTOPATHOLOGICAL OUTCOMES IN MALE

RATS FOLLOWING SPINAL CORD CONTUSTION INJURY

5.1 Introduction

Impairment of autonomically mediated functions, such as urogenital tract

function, following spinal cord injury (SCI) and its subsequent complications are

highly prevalent and clinically very important (Hicken et al., 2001; Noreau et al.,

2000). Detrusor-sphincter dyssynergia (DSD), cystitis, vesicoureteral reflux,

pyelonephritis, renal stone formation, bladder cancer, and impotency are some of

the disorders that arise due to lack of normal micturition and sexual function.

Although improvement in bladder care of spinal cord injured people over the years has led to a reduction of bladder disorders and death due to renal disease

(Ku et al., 2005), urologic care continues to be a challenge. In a recent survey amongst paraplegic and quadriplegic people, improving bladder and bowel

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function and regaining sexual function were considered most important for quality

of life (Anderson, 2004).

Innervation of the eliminative and sexual organ systems consists of both

autonomic and somatic components. In most species, motor neurons that

innervate the anal and urethral sphincters, bulbospongiosus and ischiocavernosus muscles, are all located in one cell group referred to as Onuf’s

nucleus. In rats, homologues of this nucleus are the dorsomedial (DM) and

dorsolateral (DL) nuclei located within the L5-L6 spinal cord (Breedlove and

Arnold, 1980; McKenna and Nadelhaft, 1986; Vera and Nadelhaft, 1992). Loss of

autonomic regulation of eliminative and sexual functions following SCI results

from disruption of ascending afferent pathways in the spinal cord and disruption

of descending input to the lumbosacral and sacral spinal cord. Recently, we developed a method to assess recovery of micturition and erectile function in conscious freely moving rats by monitoring corpus spongiosum pressure (CSP) by telemetry (Nout et al., 2005). Using this technique, we demonstrated that following incomplete SCI, recovery of micturition occurs in rats; however, this recovery is limited and voiding remains inefficient. Moreover, changes consistent with the development of spasticity and/or hyperreflexia were determined through waveform analysis. This is consistent with the long-term changes occurring in muscle EMG patterns, micturition frequency, volume, and bladder weight others have shown (Chancellor et al., 1994; Cheng et al., 1999; Kruse et al., 1993;

Yoshiyama et al., 2000; Yoshiyama et al., 1999).

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In order to repair the injured spinal cord, research is focused on reducing

secondary degeneration and promoting regeneration. The adult rat spinal cord

has an endogenous progenitor cell population that, according to some studies, is

mainly located in the ependymal region around the central canal (Beattie et al.,

1997; Mothe and Tator, 2005, Namiki and Tator, 1999), and according to others

is present throughout the spinal cord (Horner et al., 2000). The response of these cells after injury, however, is insufficient and does not lead to adequate recovery following SCI (Beattie et al., 1997; Horner et al., 2000; Mothe and Tator, 2005;

Namiki and Tator, 1999). Explanations that have been presented for this

insufficient endogenous response include insufficient numbers of these cells, the

hostile milieu of the lesion center, lack of neurotrophic stimulation and absence of

permissive substrates, and presence of inhibitory factors; these have been

reviewed by others (Cao et al., 2002b; Horner and Gage, 2000; Popovich et al.,

1999; Schmidt, C. E. and Leach, 2003). Cell transplant strategies have the

potential of reducing secondary damage and promoting regeneration through

many mechanisms such as replacement of lost cells and production of

permissive substrates for regeneration (Cao et al., 2002a; McDonald et al., 2004;

Okano, 2002; Zhang, 2003). At this time, it appears that optimizing the function of

neuronal circuits that survive SCI is the most achievable strategy (McDonald et

al., 2004), for example through enhancement of glial cell function.

Oligodendrocytes and astrocytes are the major supportive cells within the central

nervous system and since they are responsible for myelination of axons and

production of trophic factors in addition to providing a surface for axonal growth,

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it is reasonable to hypothesize that replacement of this cell population will positively affect regenerative processes.

Immature glial cells have been shown to reduce the inhibitory character of the lesion environment and promote axonal regeneration (Hill et al., 2004; Houle and Reier, 1988; Reier and Houle, 1988; Smith and Silver, 1988). Immature oligodendrocytes provide remyelination after injury (Kohama et al., 2001;

McTigue et al., 2001), whereas immature astrocytes promote axonal growth and perhaps promote survival after injury (Silver, 1993). A recent study supports the idea of ensuring both of these cell types, oligodendrocytes and astrocytes, are replaced, since oligodendrocyte precursors failed to remyelinate the spinal cord in the absence of astrocytes (Talbott et al., 2005). After transplanting pluripotent stem cells into normal or lesioned adult rat spinal cord most of these cells had differentiated into astrocytes and no neurons or oligodendrocytes were seen

(Cao et al., 2001). This suggests that it may be necessary to induce progenitor cells to differentiate into a certain lineage prior to transplantation. Studies have shown beneficial effects of transplanting glial progenitor cells into the injured spinal cord both in terms of behavior and histopathology (Han et al., 2004;

Keirstead et al., 2005; Lee et al., 2005). Our laboratory has previously shown that glial restricted precursor (GRP) cells derived from transgenic rats harboring the heat-stable human placental alkaline phosphatase gene (Kisseberth et al., 1999;

Rao et al., 1998) survive and differentiate when transplanted into the spinal cord immediately following injury. Also, under those conditions, GRP cells altered the

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lesion environment through reducing astrocytic scarring and through reduced expression of inhibitory proteoglycans (Hill et al., 2004).

Since other studies have shown that the optimal time window for transplanting cells into the lesion site is around day 9 after SCI (Ogawa, Y. et al.,

2002b; Okano, 2002), for the current study we use a delayed transplant strategy.

Additionally, evaluation of most transplant studies indicates that for optimal results it seems necessary to add substances that promote repair or reduce inhibitors to these cell transplantation techniques. In the current study we elevate cyclic adenosine monophosphate (cAMP) concentrations since this has recently been shown to have beneficial effects on axonal regeneration following SCI (Gao et al., 2004; Nikulina et al., 2004; Pearse et al., 2004; Spencer and Filbin, 2004).

The present study evaluates the effects of GRP cell transplantation and elevation of cAMP concentrations on autonomic outcome measurements and histopathological features. More specifically, we used chronic telemetric monitoring of CSP pressure in awake, freely moving rats to determine recovery of both micturition and erectile events following different interventions including delayed transplantation of GRP cells and elevation of cAMP concentrations through systemic phosphodiesterase IV (PDE-IV) inhibitor and local dibutyryl cAMP (db-cAMP) administration after a moderate spinal cord contusion injury.

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5.2 Materials and Methods

Study Design

Fifty-two adult, male Long-Evans hooded rats (Simonsen Laboratories,

Gilroy, CA, USA) were used in this study; forty-six rats were age 71 ± 2 days

(mean ± SE) and weighed 293 ± 1 g and 6 rats were approximately age 155 days and weighed 469 ± 14 g. This last group served as an end-point age-matched uninjured control (AM control) group for collection of telemetric and histopathological data (Table 5.1). One rat (age 71 days) was used to verify one of the immunohistochemical staining protocols.

Forty-five rats were divided in 4 groups (Table 5.1) in order to investigate the effects of GRP cell transplantation and elevation of cAMP concentrations following SCI. The operated control (OP control) group consisted of 11 rats; these rats were subjected to SCI, received 0.45% NaCl in dimethyl sulfoxide

(DMSO) subcutaneously (SQ) by osmotic pump for 14 days, and received

3x3.3μl phosphate buffered saline (PBS, pH=7.4) injected into 3 sites of the lesion region and 2x0.25μl 0.7% NaCl at 0.5cm rostral and 0.5cm caudal to the lesion center. The GRP control group consisted of 11 rats that received GRP cells only; these rats were subjected to SCI, received 0.45% NaCl in DMSO SQ by osmotic pump for 14 days, and received 2-3x106 GRP cells in 10μl PBS divided into 3 sites in the lesion region and 2x0.25μl 0.7% NaCl at 0.5cm rostral and 0.5cm caudal to the lesion center. The cAMP control group consisted of 12 rats that received cAMP only; these rats were subjected to SCI, received the

PDE-IV inhibitor rolipram (0.5mg/kg/day) in DMSO SQ by osmotic pump for 14

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days, and received 3x3.3μl PBS into 3 sites of the lesion region and 2x0.25μl

50mM db-cAMP at 0.5cm rostral and 0.5cm caudal to the lesion center. Eleven rats were administered GRP cells and cAMP (GRP cAMP); these rats were subjected to SCI, received rolipram (0.5mg/kg/day) in DMSO SQ by osmotic pump for 14 days, and received 2-3x106 GRP’s in 10μl PBS divided into 3 sites in

the lesion region and 2x0.25μl 50mM db-cAMP at 0.5cm rostral and 0.5cm

caudal to the lesion center.

AM control OP control GRP control cAMP control GRP cAMP

Age (days) 155 71 71 71 71

Total n 6 11 11 12 11

SCI, SCI, SCI, SCI,

PBS, GRP’s, PBS, GRP’s, Intervention None NaCl, NaCl, db-cAMP, db-cAMP,

NaCl NaCl rolipram rolipram

Transducer n 5/6 7/11 7/11 7/12 9/11

Histology n 6/6 10/11 10/11 11/12 9/11

Transverse N/A 5/10 5/10 6/11 5/9

Longitudinal N/A 5/10 5/10 5/11 4/9

Lumbo-sacral 6/6 8/10 10/10 11/11 9/9

Table 5.1: Outline of experimental groups. For each group age, total number (n), and interventions are shown. Furthermore, the number of animals with transducers and the number of animals that were available for histopathology are shown. Also, the number of animals from which the lesion center was sectioned transversally versus longitudinally is given. AM control = age-matched control, OP control = operated control.

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Rats were housed individually in plastic cages, maintained on a 12-h

light/dark cycle, and had free access to food and water. All animal experiments

were conducted after approval by the Institutional Laboratory Animal Care and

Use Committee of The Ohio State University and were performed in compliance

with NIH guidelines and recommendations.

Figure 5.1: Time line for surgical manipulations and administration of treatments. SQ = subcutaneous, IP = intraperitoneal.

Surgical Procedures and Post-Operative Care

Surgical procedures (Figure 5.1) were carried out aseptically under deep

anesthesia. Anesthetic plane was determined by withdrawal to foot pinch.

Lacrilube ophthalmic ointment (Allergan Pharmaceuticals, Irvine, CA, USA) was

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applied to the eyes prior to surgery and body temperature was maintained at

37.5 ± 0.5ºC using a rectal thermal probe and heating pad. For all surgical

procedures, a pre-operative dose of cefazolin (Ancef, Abbott Laboratories, North

Chicago, IL, USA; 50 mg/kg SQ) was administered.

Transducer implantation: Transducers were implanted in 7 out of 11 OP control

rats, 7 out of 11 GRP control rats, 7 out of 12 cAMP control rats, 9 out of 11 GRP

cAMP rats, and 5 out of 6 AM control rats (Table 5.1). Anesthesia was induced

by inhaled isoflurane (IsoFlow, Abbott Laboratories, North Chicago, IL, USA; 2-

3%). For transducer implantation the rat was placed in dorsal recumbency and

surgical sites were shaved and cleaned with betadine. A 3 cm skin incision was

made on midline on the raphe scroti and a 3 – 4 cm skin incision was made

parallel to midline in the right inguinal area. A telemetric pressure transducer

catheter (TA11PA-C40, Data Sciences International, St. Paul, MN, USA) was

implanted in the bulb of the CSP as described previously (Nout et al., 2005;

Schmidt, M. H. et al., 1995). Briefly, the battery of the transducer was placed

subcutaneously in the lower right abdomen and secured to the external oblique abdominal muscle with 2 sutures. The catheter was tunneled subcutaneously to the perineal area. Testes were retracted and bulbospongiosus muscle and

corpus spongiosum penis (CSP) was exposed. A guide hole was made into the

CSP with a 21 gauge needle and the open tip of the catheter was placed into the

bulb of the CSP and secured with biological glue and 1 suture. The scrotal

incision was then closed in 1 layer and the inguinal incision was closed in 2

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layers. The rat was allowed to recover. Telemetric studies commenced 4 – 5

days post operatively. Rats had access to 20ml/day of water containing maple

syrup (60ml/L).

Spinal cord injury and osmotic pump placement: Anesthesia was induced by

intraperitoneal (IP) administration of pentobarbital (Abbott Laboratories, Chicago,

IL, USA; 50 mg/kg). On “day 0”, 6 days following transducer implantation, a 25 g-

cm spinal cord contusion injury (SCI) was delivered with a MASCIS/NYU device

as previously described (Gruner, 1992). Briefly, a dorsal midline incision was

made and a dorsal laminectomy at thoracic vertebra (T)9 –T10 was performed.

The spinal cord, with an intact dura mater, was impacted with a 10 g rod from a

height of 25 mm. After closing the muscle layers, an Alzet® osmotic pump (model

2ml2; Durect Corporation, Cupertino, CA, USA) was placed subcutaneously to deliver either rolipram (Sigma, St. Louis, MO, USA; 0.5mg/kg/day) in DMSO

(Sigma, St. Louis, MO, USA; cAMP control and GRP cAMP groups) or 0.45%

NaCl in DMSO (OP control and GRP control groups) for 14 days. These pumps were primed overnight and release a set volume of 5µl/hour. The skin was apposed with skin staples and the animal was allowed to recover.

Post-operative care included continued administration of cefazolin SQ for

12 days, and once or twice daily animal check and bladder expression as needed

throughout the remainder of the study. Furthermore, animals were administered

lactated Ringer’s solution SQ (Abbott Laboratories, North Chicago, IL, USA) if

there was evidence of dehydration or hematuria, and nutritional supplementation

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in the form of Nutri-Cal (Evsco Pharmaceuticals, Division of Vétoquinol USA Inc.,

Buena, NJ, USA) if animals lost more than 10% of bodyweight.

Transplantation: On days 9 and 10 after SCI, rats were administered cyclosporin

(Bedford Laboratories, Bedford, OH, USA; 5mg/kg/day IP) as described by Ibarra et al. (1996 a,b). On day 9 after SCI animals were anesthetized with inhaled isoflurane (2-3%). A pre-operative dose of buprenorphine hydrochloride

(Buprenex, Reckitt Benckiser Pharmaceuticals Inc., Richmond, VA, USA;

0.05mg/kg SQ) was administered. The dorsal midline incision was re-opened, the injured spinal cord was re-exposed, and the rostral half lamina of T11 was removed. The transplant procedure was carried out using a 10μl bevel-tip

(diameter = 180 μm) FlexiFil microsyringe (World Precision Instruments Inc.,

Sarasota, FL, USA) assembled in a micromanipulator. The first 3.3μl of either a

GRP cell suspension (2-3x106 cells) or PBS was injected over 3 min in the lesion center. The second and third 3.3μl aliquots were injected into the lesion region at

3.5 ± 0.1 mm rostral and distal to the first injection site. Depth of injections was approximately 1 mm. Using a similar 10μl FlexiFil syringe assembled in the micromanipulator, 0.25μl db-cAMP (Sigma, St. Louis, MO, USA; cAMP control and GRP cAMP groups) or NaCl (OP control and GRP control groups) was administered on either side (0.7mm) of midline approximately 1mm rostral and caudal to the rostral and caudal injections sites, respectively. Depth of injections was 1 mm. The site of injections is shown in Figure 5.2. The muscle layers were

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sutured and the skin was apposed with skin staples. The animal was allowed to

recover from anesthesia.

On day 11 after SCI the maple syrup water was replaced by 20ml/day of

1.2% liquid vitamin C (Vedco, Inc., St. Joseph, MO, USA) prepared in maple

syrup water (60ml/L). Also, on day 11 after SCI cyclosporin was added to the drinking water. Our goal was to deliver 10mg/kg/day by mouth of cyclosporin throughout the remainder of the study duration (Ibarra et al., 1996a; Ibarra et al.,

1996b). For this we assumed that our rats drank 120-144ml/day.

Figure 5.2: Site of injections made into the dorsal spinal cord during the transplant procedure. Thick arrows indicate the site of the 3 GRP or PBS injections. Thin arrows indicate the site of the 4 db-cAMP or NaCl injections.

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Osmotic pump removal: On day 14 after SCI rats were anesthetized with inhaled isoflurane. The skin at the dorsal midline incision was re-opened over a length of

1cm and the osmotic pump was removed. The skin was apposed with skin staples and the animal was allowed to recover from anesthesia.

Sacrifice: At 12 weeks post SCI animals were anesthetized with xylazine

(TranquiVedTM, Vedco Inc., St. Joseph, MO, USA; 10 mg/kg IP) and ketamine

(ketamine HCl, Abbott Laboratories, N.Chicago, IL, USA; 80 mg/kg IP). This was

followed by transcardiac perfusion with 0.9% NaCl and 4% paraformaldehyde in

PBS. The lesion region and the L4-S2 segment of the spinal cord were isolated

and kept in 4% paraformaldehyde for 24 hours and cryoprotected in 30% sucrose

in PBS for 24-48 hours. The tissue was frozen at –80ºC until further analysis.

Glial Restricted Precursor Cells

Transgenic E13.5 rat embryos were used that harbored the heat-stable

human placental alkaline phosphatase (hPLAP) gene. In these transgenic rats,

the ROSA26 gene promoter directs ubiquitous expression of the hPLAP marker gene (Kisseberth et al., 1999). Glial restricted precursor (GRP) cells were isolated as described previously (Hill et al., 2004; Rao et al., 1998). Trunk

segments including the last 10 somites were dissected and titrated to remove the

neural tube from the somites. The neural tube was dissociated using papain

digestion. E-NCAM-positive cells, which have been shown to be neuron-

restricted precursor cells (Mayer-Proschel et al., 1997), were removed by plating

the suspension onto E-NCAM-coated dishes for 20 min at 37ºC. The supernatant

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was then removed and A2B5-positive GRP cells were isolated by positive

immunopanning. Cells were plated onto A2B5 antibody-coated dishes for 20 min

at 37ºC. The supernatant was removed, the plate washed with culture medium,

and the bound A2B5-positive cells were placed into 75 cm2 tissue culture flasks,

coated with fibronectin/laminin solution (FN/LN) containing DMEM-F-12-BS with

10μg/ml of bFGF (10ml total/75 cm2 flask). Cells were grown in an incubator at

37ºC, 6-7.5% CO2. After 5-7 days, when cultures reached 50-70% confluence,

cells were harvested and transported overnight for transplantation. This

procedure yields >98% A2B5-positive cells (Hill et al., 2004). Previous clonal

studies have confirmed that these A2B5-positive cells are all GRP cells and can

differentiate into oligodendrocytes and astrocytes but not neurons (Mayer-

Proschel et al., 1997; Rao et al., 1998).

On arrival, prior to transplantation, viability and number of GRP cells were

assessed. The suspension (15 ml total volume) was centrifuged (Centra CL3R,

International Equipment Company, Needham Heights, MA, USA) at 300g

(1000rpm) for 5 min at room temperature. The pellet containing the GRP cells

was then re-suspended in 10μl PBS and used for transplantation as described

above.

Behavioral Tests

Locomotor: Open-field walking was evaluated before SCI (Baseline, BL) and at

24 hours, 48 hours, and 7, 10,16, 22,30, 37, 44, 51, 58, 65, 72, 79, and 86 days

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following SCI using the 21-point BBB Locomotor Rating Scale (Basso et al.,

1995). Furthermore, activity in the cage was determined by telemetry. Movement

of the transducer over different detection fields of the receiver was recorded and

using Dataquest ART software (Data Sciences International, St. Paul, MN, USA)

a moving average in counts per minute was acquired. Data were recorded on

days 1, 5-8, 10-15, 21, 28, 35, 42, 49, 56, 63, 70, 77, and 84 after SCI.

Micturition and erectile function: Telemetric data were collected on 7 out of 11

OP control rats, 7 out of 11 GRP control rats, 7 out of 12 cAMP control rats, 9 out of 11 GRP cAMP rats, and 5 out of 6 AM control rats (Table 1). Micturition and erectile characteristics were evaluated as previously described (Nout et al.,

2005). Briefly, physiological telemetry data (CSP pressure) were recorded on a

PC computer using Dataquest ART software. Data were subsequently analyzed with Spike2 data acquisition and analysis software (version 3.1, Cambridge

Electronic Design Limited, Science Park, Milton Road, Cambridge CB40FE,

U.K.). All recording occurred while animals were housed in their regular cages.

Animals underwent 2x24h CSP pressure baseline (BL) recording prior to SCI.

After SCI, CSP pressure was recorded (24 hours/day) on days 1 – 15, 21, 28, 35,

42, 49, 56, 63, 70, 77, and 84 and analyzed for days 2, 5, 14, and 84.

CSP pressure data were analyzed for the total number of micturitions, full

erectile events (tumescence pressure of at least 30 mmHg above baseline on top

of which at least 1 suprasystolic pressure peak of 100 mmHg above the

tumescence pressure occurs), and partial erectile events (similar to full erectile

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events with the exception that peaks never reach 100 mmHg over the

tumescence pressure) in a 24 hour period. Furthermore, a detailed waveform

analysis was performed for 4 days (BL, day 5, 15, 84). For micturitions and

partial erectile events this was performed on the complete 24 hour period and for

full erectile events this was performed on an 8 hour window (9pm – 5am) within

the 24 hour recording period. Data analyzed were duration of the events, area

under the curve (AUC), total number of pressure peaks, peak frequency and

mean and maximum pressures. Furthermore, a peak analysis was performed.

For micturition events this included the number of afterpeaks, and afterpeak

duration, AUC, and mean and maximum pressures. For erectile events this

included the duration, AUC, and mean and maximum pressures of the peaks that

occurred during those events.

Reflex erection tests: Ex copulatory reflex erection tests were performed as

previously described (Hart, 1968; Nout et al., 2005; Schmidt, M. H. et al., 1995).

Briefly, rats were placed on a board in dorsal recumbency with the head and anterior torso in a loose-fitting restraining cylinder. The abdomen was secured to the board with masking tape. The head and anterior torso could move freely in the cylinder. The telemetric receiver unit was placed directly under the board.

The preputial sheath was retracted, which is the stimulus to elicit reflex erections, and maintained in retracted position by placing the glans penis through a hole in a small piece of masking tape fastened to the abdomen. Once the sheath was retracted, the reflex test lasted for 20 min. Rats were habituated to this procedure

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prior to surgery for 2x10 min and 2x20 min and the animals quickly adapt to the

testing situation. Events were visually scored according to previously described

criteria (Schmidt, M. H. et al., 1995): E1, weak glans engorgement; E2, moderate

glans engorgement involving some dilation of the distal glans; E3, intense flaring

or cup of the distal glans; F1, dorsiflexion of the penile body; F2, dorsiflexion or

flip of the penile body greater than 90º with respect to the body of the rat. These

reflexes were scored with a numerical keypad as events 1 – 5, respectively.

Reflex testing was performed twice prior to SCI and on days 2, 7, 16, 22, 30, 44,

58, 72, and 86 following SCI. The 6 animals in the AM control group were tested

twice, once prior to and once after transducer implantation.

Histopathology and Analysis – Lesion Center

Lesion regions (10mm) were sectioned at 20 μm on a cryostat

transversally (5 out of 10 OP control animals, 5 out of 10 GRP control animals, 6

out of 11 cAMP control animals, and 5 out of 9 GRP cAMP animals) or

horizontally (5 out of 10 OP control animals, 5 out of 10 GRP control animals, 5

out of 11 cAMP control animals, and 4 out of 9 GRP cAMP animals) (Table 5.1).

Lesion regions were randomly assigned to each of the 2 groups.

First, we verified that the immunohistochemical staining protocol using the human placental alkaline phosphatase (hPLAP) antibody described below was

useful in detecting the transplanted GRP cells in vivo. One animal was sacrificed

48 hours after receiving an injection of 3x106 GRP cells into the spinal cord at T9.

The technique used for injecting GRP cells was similar to that described for the

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transplantation surgery. The injection area was cut transversally at 20 μm on a

cryostat. Every other section was stained for hPLAP using both the enzymatic

alkaline phosphatase (AP) staining method and the immunohistochemical protocol described below and in Table 5.2.

Endogenous AP was denatured by heating the slides to 60ºC for 1 hour

before reacting the tissue with Nitro blue tetrazolium chloride/5-bromo-4-chloro-3-

indolyl phosphate (NBT/BCIP, toluidine salt; Roche Diagnostics Corporation,

Indianapolis, IN, USA; NBT/BCIP stock solution diluted 1/20) for 1.5 hours in AP

buffer (100 mM Tris, 100 mM NaCl, 50 mM MgCl2, pH 9.5) with 5mM levamisole

to block any remaining endogenous AP activity. The other sections were stained

using the immunohistochemistry procedure described below.

Immunohistochemistry: Every sixth transverse section was stained for hPLAP

and glial fibrillary acidic protein (GFAP, for astrocytes), hPLAP and CC-1 (for

oligodendrocytes), and hPLAP and nestin (for undifferentiated cells). Every third

horizontal section was stained for hPLAP and serotonin. Immunohistochemistry was performed at room temperature. Slides were first rinsed with PBS for 2x10 min and subsequently incubated with blocking solution containing 10% Goat

Serum (Sigma, St. Louis, MO, USA) and 0.2% triton in PBS for 60 min.

Incubation with primary and secondary antibodies followed as specified in Table

5.2 after which slides were rinsed with PBS for 2x10 min. Slides were then cover- slipped with Vectashield with DAPI (nuclear counterstain) hard-set mounting

media (Vector’s Laboratory Inc., Burlinghame, LA, USA and stored at 4ºC.

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Primary antibody Secondary antibody Buffer

Inc. Inc. Name [ ] Isotype Source Name [ ] Source time time

Invitrogen, AF-594 1:200 2 hours Carlsbad, PBS Mouse Sigma, St. GAM IgG2a hPLAP 1:200 O/N CA monoclonal IgG2a Louis, MO AF-488 1:200 2 hours Invitrogen PBS GAM IgG

Abcam Mouse AF-488 CC-1 1:100 O/N Cambridge, 1:200 2 hours Invitrogen PBS monoclonal IgG2b GAM IgG2b MA

Rabbit polyclonal AF-488 GFAP 1:1000 O/N Sigma 1:200 2 hours Invitrogen PBS IgG GAR IgG

Mouse hybridoma DSHB, Iowa AF-488 Nestin 1:10 O/N 1:200 2 hours Invitrogen PBS supernatant IgG1 City, IA GAM IgG1

Rabbit polyclonal Immunostar, AF-488 5-Ht 1:500 O/N 1:200 2 hours Invitrogen PBS IgG Hudson, WI GAR IgG

Chemicon, Mouse AF-546 Syn. 1:200 O/N Temecula, 1:200 2 hours Invitrogen PBS monoclonal IgG1 GAM IgG CA

Table 5.2: List of antibodies used for immunohistochemistry. Source, concentration, and incubation time is shown for each antibody. Inc. time = incubation time, The Nestin Rat-401 hybridoma was developed by Susan Hockfield and obtained from the Developmental Systems Hybridoma Bank (DSHB) developed under the auspices of the NICHD and maintained by the University of Iowa, Department of Biological Sciences, Iowa City, IA, USA. [ ] = concentration, Inc. time = incubation time, hPLAP = human placental alkaline phosphatase, O/N = overnight, CC-1 = APC antibody CC-1, GFAP = glial fibrillary acidic protein, 5-Ht = serotonin, Syn. = synaptophysin, GAM = goat-anti-mouse, GAR = goat-anti-rabbit, AF = Alexa-Fluor, PBS = phosphate buffered saline (ph=7.4).

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Analysis: For the 21 animals from which the lesion region was cut transversally,

the area of tissue at the lesion center was determined using Adobe Photoshop vs.5 (Adobe Systems Incorporated, San Jose, CA, USA). For this, one section per animal taken at the lesion center was used. These sections were stained for hPLAP and GFAP. Slides were examined and images taken under standard fluorescence at 10x (Zeiss Axioplan2 microscope, Carl Zeiss, Hallergmoos,

Germany). Dimensions were normalized to a more distal section to correct for

tissue loss. We determined GFAP and hPLAP area in one section at the lesion

center that was identified by containing the smallest amount of tissue.

For the 21 animals that received a GRP cell transplant, an approximation

of the volume of GRP cells that occupied the lesion region was determined. In

the transversally cut sections this was done by first imaging every other section

on slides stained for hPLAP and GFAP. Images were then analyzed using

MetaMorph software. The outline of the section, lesion cavity, and hPLAP-

positive area were traced. Area of lesion cavity was subtracted from the section

area and the percentage of hPLAP-positive area was calculated for each animal.

The area of GRP cells in horizontal sections was determined in sections stained

for hPLAP and serotonin using PhotoShop. For the 9 animals in this group 3

images of each lesion region were made. Area of tissue, hPLAP-positive area,

and area of lesion cavities were determined. The percentage hPLAP-positive

area in the lesion region was determined.

To determine GRP cell survival and differentiation we examined sections

for the presence of double-labeled cells expressing hPLAP and nestin, hPLAP

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and GFAP, or hPLAP and CC-1. Slides from 10 animals were examined and images were taken on a Zeiss 510 META confocal laser scanning microscope

(Hallergmoos, Germany) at 63x. Stacks of images were collected with a distance of 0.37 μm between consecutive slices. These stacks were processed using

AutoDeblur & AutoVisualize vs.9.3 software (AutoQuant Imaging Inc., Watervliet,

NY, USA) and analyzed using MetaMorph vs.6.3 software (Molecular Devices

Corporation, Downingtown, PA, USA). A rostral, middle, and caudal section were used for this analysis. Images were made from the area where the transplant integrated with the host tissue. Three images were made from 3 different parts of the transplant in that section and while choosing the area to be imaged, the slide was examined for hPLAP only. This ensured our sampling technique was not based on double-labeling. The slice showing most co-localization was determined using MetaMorph software and cells were manually counted. The number of cells that expressed hPLAP was determined and the number of cells that expressed both hPLAP and nestin, hPLAP and CC-1, or hPLAP and GFAP was determined.

Histopathology and Analysis – Lumbosacral Spinal Cord

Lumbosacral spinal cord blocks for all available animals (n = 44) were sectioned transversally at 20 μm on a cryostat. In order to evaluate the serotonergic input to the lumbosacral spinal cord, immunohistochemistry for synaptophysin and serotonin was performed on every other section. The immunohistochemical protocol was similar to what is described above and in

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Table 5.2 for the lesion region, with the exception that TOTO-3 cyanine dimer

nucleic acid stain (Invitrogen Corporation, Carlsbad, CA, USA; 2mM) was added

to the solution containing the secondary antibodies. Slides were then cover-

slipped with Vectashield hard-set mounting media (Vector’s Laboratory Inc.,

Burlinghame, LA, USA and stored at 4ºC.

Analysis: Slides from 44 animals were examined and images taken on a Zeiss

510 META confocal laser scanning microscope (Hallergmoos, Germany) at 63x

with 2x zoom. For each animal 12 motor neurons located in the DL nuclei (6 left

side, 6 right side) and 8 motor neurons located in the DM nuclei (4 left side, 4

right side) were imaged. Images were made based on synaptophysin and TOTO-

3 staining. Serotonin staining was not examined prior to collecting the images.

Stacks of images were collected with a distance of 0.37 μm between consecutive slices. These stacks were processed as described above using AutoDeblur &

AutoVisualize vs.9.3 software and analyzed using MetaMorph vs.6.3 software.

The amount of serotonin within the stack was determined. This enabled us to quantify the amount of serotonin in close proximity to motor neurons in the DM and DL nuclei.

Statistics

Data are presented as means ± standard error of the mean (SE). A factorial repeated measures analysis of variance (ANOVA) was used to analyze

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all behavioral data. Furthermore, a factorial repeated measures analysis of

covariance (ANCOVA) with the covariate factor being baseline data was used to analyze behavioral data. The null hypothesis was rejected at α = 0.05. A one-way

ANOVA was used to compare BL and end point data of the OP control, GRP control, cAMP control, and GRP cAMP groups to that of the AM control group. A one-way repeated measures ANOVA was used to determine significant differences between BL and endpoint data. Significant differences identified by the ANOVA were isolated using the Holm-Sidak procedure for pairwise multiple comparison post-hoc test.

A t-test was used to analyze the area of tissue occupied by GRP cells and

the differentiation of the GRP cells. Other histopathological data were analyzed

using a one-way ANOVA. Significant differences identified by the ANOVA were

isolated using the Holm-Sidak procedure for pairwise multiple comparison post-

hoc test. The statistical computations were performed with software packages

(Sigmastat 3.0, SPSS, Chicago, IL and SPSS 12.0, SPSS, Chicago, IL).

5.3 Results

Rats were 71 ± 2 days and weighed 293 ± 1 g for transducer implantation.

Rats were 77 ± 2 days and weighed 318 ± 2 g at the time of SCI. At the time of transplant and sacrifice rats weighed 289 ± 3 g and 385 ± 6, respectively. At the time of sacrifice average weights of animals were similar in all groups (OP control: 392 ± 11 g; GRP control: 383 ± 14 g; cAMP control: 380 ± 13 g; GRP cAMP: 391 ± 12 g). Five animals died unexpectedly during the time-course of this

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study; 1 animal from the OP control group died on day 9 following SCI, 1 animal

in the cAMP control group died on day 11 following SCI, 1 animal in the GRP

control group died on day 22 following SCI, and 2 animals in the GRP cAMP

group died on days 53 and 79 following SCI.

The moderate to severe SCI that rats were subjected to in this study was

a very consistent lesion based on the parameters recorded by the impactor

device (Figure 5.3).

Impact parameters

2.5

1.5 OP control GRP control cAMP control Value 1 GRP cAMP

0.5

0 Velocity (m/s) Compression (mm) Compression rate (m/s)

Figure 5.3: Impact parameters. Subset of impact parameters shown for the 4 groups of rats injured in this study.

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Behavioral Outcomes

Locomotor: The 25 g-cm SCI resulted in an initial profound paraplegia in all rats

characterized by BBB locomotor scores of 0.6 ± 0.3 (OP control) 0.5 ± 0.3 (GRP

control) 0.4 ± 0.3 (cAMP control), and 0.6 ± 0.1 (GRP cAMP) at 24 hours after

SCI. Gradually, rats showed some recovery of hind limb locomotor function

(Figure 5.4), however, no significant differences were found between groups.

BBB locomotor scores were 8.2 ± 0.4 (OP control) 8.0 ± 0.6 (GRP control) 8.0 ±

0.5 (cAMP control), and 8.6 ± 0.4 (GRP cAMP) at the time of sacrifice (Figure

5.4). This remained significantly different from BL (p<0.001; F=404). A BBB score

of 8 indicates animals are able to extensively move all 3 joints of the hind limbs,

and in some cases animals are able to place their paws in a plantar position,

however, there is no weight support.

The level of activity of the rats when housed in their cages is shown in

Figure 5.5. The immediate reduction of activity following SCI was followed by a

steady recovery during the first 3 weeks after SCI. The transplantation surgery at

day 9 resulted in a second drop in level of activity. This is seen in Figure 5.5 as a

set-back during the recovery phase after SCI. In our animals the level of activity reached a plateau between 3 – 5 weeks following SCI. The level of activity of all rats at the end point of this study was similar to that of the AM control group,

which was significantly less than activity recorded for rats at BL for the OP

control (p=0.03; F=9), cAMP control (p<0.001; F=36), and GRP cAMP (p=0.009;

F=14) groups. No significant differences were found between groups.

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BBB locomotor scores

15 OP control GRP control 10 cAMP control GRP cAMP

5 BBB locomotor score BBB locomotor

0 BL1 2 7 101622303744515865727986 Time (day)

Figure 5.4: Recovery of locomotion function. For the 4 groups of rats the BBB locomotor score (AVG ± SE) is shown for baseline (BL) and for the duration of the study. Black arrow indicates time of transplantation surgery (day 9). All groups significantly different from BL.

Moving average

40 OP control

30 GRP control cAMP control 20 GRP cAMP Count (24 h) (24 Count AM control 10

0 BL BL 1 2 3 4 5 6 7 8 10 11 12 13 14 15 21 28 35 42 49 56 63 70 77 84 Time (day)

Figure 5.5: Activity of all rats in their cages. The level of activity is shown in arbitrary units. Black arrow indicates time of transplantation surgery (day 9). OP control, cAMP control, GRP cAMP, and AM control significantly different from BL. 129

Micturition function: Baseline data showed an average number of micturition

events per 24 hours per rat of 22 ± 2 (OP control), 20 ± 2 (GRP control), 29 ± 2

(cAMP control), and 27 ± 1 (GRP cAMP). First spontaneous micturition events

were detected by telemetry 2 – 4 days following SCI. Recovery of the total

number of micturition events is shown for BL and days 2, 5, 14, and 84 after SCI

in Figure 5.6A. Total number of micturition events on day 84 following SCI was

12 ± 3 (OP control), 10 ± 2 (GRP control), 15 ± 3 (cAMP control), and 19 ± 1

(GRP cAMP). No statistically significant differences were present between

groups. To determine the effect of baseline variation an ANCOVA was

performed. Results are plotted (Figure 5.6B).

Although in most animals recovery of micturition was sufficiently effective that manual bladder expression was no longer required, the number of micturitions per 24 hour period at the end point of the study remained

significantly lower than the number of micturitions recorded at baseline for all

groups (OP control: p=0.03; F=12; GRP control: p=0.05; F=11; cAMP control:

p=0.02; F=11; GRP cAMP: p=0.008; F=18). Furthermore, the number of

micturitions at the end point of the study was lower than the number of

micturitions recorded from the AM control group (23 ± 2) as shown in Figure

5.6A; however, this was only significant for the OP control and GRP control

groups (p=0.008; F=4.6).

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Recovery of micturition function

25 OP control 20 GRP control cAMP control 15 GRP cAMP AM control Number (24 h) Number (24 10

0 BL 2 5 14 84 Time (day)

Corrected means - recovery of micturition

OP control 15 GRP control cAMP control 10 GRP cAMP Number (24h) Number 5

0 BL 2 5 14 84

-5 Time (day)

Figure 5.6 A,B: Recovery of micturition function shown for all groups. The number of micturitions per 24 hours recorded by telemetry is shown for baseline (BL) and days 2, 5, 14, and 84 after SCI (A). In B the estimated marginal means are shown determined by ANCOVA set at BL = 25.3 micturitions/24h. significantly different from BL; significantly different from AM control.

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Performing detailed pressure waveform analysis revealed increased

duration of micturition events over time (Figure 5.7A), increased frequency of micturition pressure peaks (Figure 5.8A), and increased mean pressures during micturition events (Figure 5.9A). To determine the effect of baseline variation

ANCOVA’s were performed on each of these parameters; results are plotted in

Figures 5.7b, 5.8B, and 5.9B.

Micturition peak frequency is a robust parameter as demonstrated by

small standard errors and little variability between groups. Furthermore, the

micturition peak frequency recorded in the AM control group was not significantly different from BL data of the other 4 groups. Comparing day 84 to BL revealed significant increase of micturition peak frequency for the OP control and cAMP control groups (p=0.03; F=11.6 and p=0.01; F=12.7, respectively). At day 84 the micturition peak frequency of the cAMP control and the GRP cAMP groups were

significantly larger compared to the AM control group (p=0.02; F=3.6). No

significant effect of group over time was seen.

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Micturition duration

9.0 8.0 7.0 OP control 6.0 GRP control 5.0 cAMP control 4.0 3.0 GRP cAMP

Duration (s) 2.0 AM control 1.0 0.0 BL 5 14 84 Time (day)

Corrected means - micturition duration

10.0 9.0 8.0 7.0 OP control 6.0 GRP control 5.0 cAMP control 4.0 GRP cAMP Duration (s) Duration 3.0 2.0 1.0 0.0 BL 5 14 84 Time (day)

Figure 5.7 A,B: Duration of micturition events. The duration of micturition events is shown for baseline (BL) and days 5, 14, and 84 after SCI (A). In B the estimated marginal means are shown determined by ANCOVA set at BL = 3.4s.

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Micturition peak frequency

12.0

10.0 OP control 8.0 GRP control 6.0 cAMP control

4.0 GRP cAMP AM control

Frequency (Hz) Frequency 2.0

0.0 BL 5 14 84 Time (day)

Corrected means - micturition frequency

14.0

12.0

10.0 OP control 8.0 GRP control 6.0 cAMP control GRP cAMP 4.0 Frequency (Hz) Frequency 2.0

0.0 BL 5 14 84 Time (day)

Figure 5.8 A,B: Frequency of micturition pressure peaks. The frequency of micturition pressure peaks is shown for baseline (BL) and days 5, 14, and 84 after SCI (A). In B the estimated marginal means are shown determined by ANCOVA set at BL = 8.6 Hz. significantly different from BL; significantly different from AM control. 134

Micturition mean pressure

90.0 80.0

70.0 OP control 60.0 GRP control 50.0 cAMP control 40.0 GRP cAMP 30.0 20.0 AM control

Pressure (mmHg) Pressure 10.0 0.0 BL 5 14 84 Time (days)

Corrected means - micturition mean pressure

70.0

60.0

50.0 OP control 40.0 GRP control 30.0 cAMP control GRP cAMP 20.0 Pressure (mm Hg) (mm Pressure 10.0

0.0 BL 5 14 84 Time (day)

Figure 5.9 A,B: Mean pressure during micturition events. The mean pressure of micturition events is shown for baseline (BL) and days 5, 14, and 84 after SCI (A). In B the estimated marginal means are shown determined by ANCOVA set at BL = 32.5 mmHg.

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Erectile function: Baseline data resulted in an average number of full erectile events per 24 hours per rat of 52 ± 3 (OP control), 63 ± 4 (GRP control), 47 ± 6

(cAMP control), and 63 ± 9 (GRP cAMP). Figure 5.10A shows the change in number of events on days 2, 5, 14, and 84 after SCI. The total number of full erectile events on day 84 following SCI was 32 ± 2 (OP control), 40 ± 10 (GRP control), 30 ± 4 (cAMP control), and 37 ± 7 (GRP cAMP). This reduction in number of erectile events per 24 hours was only significant in the OP control and cAMP control groups (p=0.006; F=28 and p=0.03; F=8.7, respectively). No statistically significant differences were present between groups or between the injured groups and the AM control group (45 ± 5 erectile events per 24h). To determine the effect of baseline variation an ANCOVA was performed; results are plotted (Figure 5.10B). Performing detailed pressure waveform analysis revealed slight increases in waveform characteristics such as erectile event duration (Figure 5.11A) and erectile event maximum pressures (Figure 5.12A); however the only significant effects were an increase in maximum pressure on day 84 after SCI compared to BL in the OP control and cAMP control groups

(p=0.02; F=9.1). To determine the effect of baseline variation ANCOVA’s were performed on each of these parameters; results are plotted in Figures 5.11B and

5.12B.

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Full erectile events

90 80 70 60 OP control GRP control 50 cAMP control 40 GRP cAMP 30 AM control Number (24 h) (24 Number 20 10 0 BL 2 5 14 84 Time (day)

Corrected means - full erectile events

50 OP control 40 GRP control

30 cAMP control GRP cAMP

Number (24 h) (24 Number 20

0 BL 2 5 14 84 Time (day)

Figure 5.10 A,B: Number of full erectile events. The number of full erectile events per 24 hours recorded by telemetry is shown for baseline (BL) and days 2, 5, 14, and 84 after SCI (A). In B the estimated marginal means are shown determined by ANCOVA set at BL = 54 erectile events per 24h. significantly different from BL. 137

Full erectile event duration

40.0

35.0

30.0 OP control 25.0 GRP control 20.0 cAMP control GRP cAMP 15.0

Duration (s) Duration AM control 10.0

5.0

0.0 BL 5 14 84 Time (day)

Corrected means - erectile event duration

35.0

30.0

25.0 OP control 20.0 GRP control 15.0 cAMP control GRP cAMP

D uration (s)10.0

5.0

0.0 BL 5 14 84 Time (day)

Figure 5.11 A,B: Duration of full erectile events. The duration of full erectile events is shown for baseline (BL) and days 5, 14, and 84 after SCI (A). In B the estimated marginal means are shown determined by ANCOVA set at BL = 22.4s.

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Full erectile event maximum pressure

800

700

600 OP control 500 GRP control 400 cAMP control GRP cAMP 300 AM control 200 Pressure (mmHg) Pressure 100

0 BL 5 14 84 Time (day)

Corrected means - erectile event maximum pressure

600

500

400 OP control GRP control 300 cAMP control 200 GRP cAMP

Pressure (mmHg) Pressure 100

0 BL 5 14 84 Time (day)

Figure 5.12 A,B: Maximum pressure during full erectile events. The maximum pressure during full erectile events is shown for baseline (BL) and days 5, 14, and 84 after SCI (A). In B the estimated marginal means are shown determined by ANCOVA set at BL = 394 mmHg. significantly different from BL. 139

Baseline data resulted in an average number of partial erectile events per

24 hours per rat of 8 ± 2 (OP control), 8 ± 2 (GRP control), 7 ± 2 (cAMP control), and 13 ± 3 (GRP cAMP). Figure 5.13A shows the change in number of events on days 2, 5, 14, and 84 after SCI. The total number of partial erectile events on day

84 following SCI was 3 ± 1 (OP control), 3 ± 2 (GRP control), 3 ± 1 (cAMP control), and 3 ± 1 (GRP cAMP). Although these numbers were significantly lower than those recorded on BL for the OP control (p=0.002, F=54), cAMP control (p=0.005; F=19), and GRP cAMP (p=0.007; F=19) animals, no statistically significant differences were present between groups. Furthermore, the number of partial erectile events per 24 hours at 84 days after SCI was lower in all injured groups compared to those recorded for the AM control group (6 ± 3), but this was not statistically significant. To determine the effect of baseline variation an ANCOVA was performed; results are plotted (Figure 5.13B).

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Partial erectile events

18 16 14 OP control 12 GRP control 10 cAMP control 8 GRP cAMP 6 AM control Number (24 h) Number (24 4 2 0 BL 2 5 14 84 Time (day)

Corrected means - partial erectile events

16 14

12 10 OP control GRP control 8 cAMP control 6 GRP cAMP

Number (24 h) Number (24 4 2

0 BL 2 5 14 84 Time (day)

Figure 5.13 A,B: Number of partial erectile events. The number of partial erectile events per 24 hours recorded by telemetry is shown for baseline (BL) and days 2, 5, 14, and 84 after SCI (A). In B the estimated marginal means are shown determined by ANCOVA set at BL = 10.6 partial erectile events per 24h. significantly different from BL. 141

Reflex erection tests: Following SCI, latency (time of first erectile event following

sheath retraction) decreased at all time points (Figure 5.14). Although some

recovery occurred, latency remained significantly lower than BL for all groups

(OP control: p<0.001; F=28; GRP control: p<0.001; F=29; cAMP control:

p=0.002; F=16; GRP cAMP: p<0.001; F=61). Furthermore, latency recorded in

the AM group was significantly lower than latency measured at BL in the injured

groups (p<0.001; F=14). At day 86 after SCI, latency in the GRP control and

GRP cAMP groups remained significantly lower than that of the AM group

(p<0.001; F=14).

Reflex erection test - latency

1000

OP control GRP control cAMP control 500 GRP cAMP Time (s) Time AM control

0 BL BL 2 7 16 22 30 44 58 72 86 Time (day)

Figure 5.14: Latency during the reflex erection test. For all groups of rats the latency (time to first erection after sheath retraction) during the ex copulatory reflex erection test is shown for baseline (BL) and for the duration of the study. all groups significantly different from BL. GRP control and GRP cAMP groups significantly different from AM control.

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The number of suprasystolic pressure peaks that occurred during erectile

events increased from BL at all time points following SCI (Figure 5.15). At day 86

after SCI the number of pressure peaks per event was significantly higher than at

BL for the GRP control (p=0.002; F=28) and cAMP control (p=0.016; F=92)

groups. Furthermore, the number of pressure peaks at day 86 following SCI

remained significantly greater than that recorded in the AM control group for all

groups (p<0.001; F=7.6).

Reflex erection test - peaks per erectile event

OP control 10 GRP control cAMP control GRP cAMP Number 5 AM control

0 BL BL 2 7 16 22 30 44 58 72 86 Time (day)

Figure 5.15: Number of peaks per erectile event. For all groups of rats the number of suprasystolic pressure peaks per event during the ex copulatory reflex erection test is shown for baseline (BL) and for the duration of the study. GRP control and cAMP control significantly different from BL. all groups significantly different from AM control.

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Histopathology – Lesion Center

Specificity of the hPLAP antibody we used in this study is demonstrated in

Figure 5.16. In Figure 5.16 two transverse sections of spinal cord are shown of a

rat that had GRP cells injected into its spinal cord 48 hours prior to sacrifice. The

section in Figure 5.16A is stained using the enzymatic AP method and in Figure

5.16B GRP cells are identified by immunohistochemistry.

A B

Figure 5.16 A,B: Thoracic spinal cord stained for GRP cells 48 hours after cell transplant. Transverse sections (20 μm) of spinal cord showing the area of the dorsal funiculi just dorsal to the central canal. In A GRP cells are identified by the enzymatic alkaline phosphatase method and appear dark brown – black. In B GRP cells are identified using immunohistochemistry and appear green. Cell nuclei appear blue due to the DAPI nuclear counterstain.

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Tissue at the lesion center: No residual gray matter was observed at the lesion

center, but a thin layer of white matter remained. Tissue (GFAP positive) present

at the lesion center was determined in transverse sections of the spinal cord at

the level of the lesion center (Figure 5.18 and 5.19). Figure 5.18 shows examples

of lesion centers for all 4 groups. In Figure 5.19A the percentage of tissue present at the lesion center is shown as compared to tissue in a more distal section of normal appearing thoracic spinal cord. In Figure 5.19B the hPLAP positive area in the lesion center is shown as a percentage of tissue in a more distal section of normal appearing thoracic spinal cord.

Figure 5.17: Illustrations of lesion center and lesion region. The lesion region typically spanned a length of 10mm, whereas the lesion center was a 20μm thick section.

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C D

Figure 5.18 A-D: Representative lesion centers for all groups. A = OP control, B = GRP control, C = cAMP control, D = GRP cAMP. Sections are 20 μm thick and stained for GFAP, hPLAP, and DAPI by immunohistochemistry. Green represents astrocytes (GFAP), red / yellow represents GRP cells (hPLAP), and blue represents nuclei (DAPI). Scale bar = 20mm.

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Tissue at lesion center normalized for tissue loss

25 OP control 20 GRP control

15 cAMP control GRP cAMP 10 Percentage (%)

hPLAP positive area at lesion center normalized for tissue loss

12 OP control 10 GRP control cAMP control 8 GRP cAMP

Percentage (%) Percentage 6

Figure 5.19A,B: Tissue at lesion center. In A the area of GFAP positive tissue at lesion center is shown as a percentage of GFAP positive tissue in a distal section of thoracic spinal cord. In B the area of hPLAP positive tissue at lesion center is shown as a percentage of GFAP positive tissue in a more distal section of thoracic spinal cord. significantly different from GRP control. 147

Although there appears to be more tissue at the lesion center in the 2 groups of animals that received GRP cell transplants, these differences were not statistically significant. The hPLAP area at the lesion center was significantly larger in the GRP control group compared to the GRP cAMP group (p<0.001;

F=15.6).

Volume of tissue and GRP cells in lesion region: GRP cells were present at 84 days after SCI in all animals that had received transplants. In the Appendix representative images are shown of the transplants of each of the 19 animals that received GRP cell transplants. Images are shown from an area in the lesion region, adjacent but not at lesion center. To determine the area occupied by tissue (GFAP positive) and GRP cells (hPLAP positive) in the transversally cut spinal cords from 10 animals, 35 – 45 (range) sections were imaged per animal.

The distance between consecutively imaged sections was approximately 200μm.

Figure 5.20 shows that there was significantly more tissue present throughout the lesion region in animals that received a GRP cell transplant (p=0.003; F=6.9).

Figure 5.21A shows the hPLAP positive area throughout the lesion region determined for the 2 groups. Although the hPLAP area in the GRP control group appears larger than in the GRP cAMP group this difference was not statistically significant. For 9 animals the hPLAP positive area was determined in horizontal sections. The distance between the 3 horizontal sections that were imaged was approximately 280 μm. The hPLAP positive area is shown for both groups in

Figure 5.21B.

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Tissue in lesion region corrected for tissue loss

50 OP control 40 GRP control cAMP control 30 GRP cAMP

Percentage (%) 20

Figure 5.20: Tissue in lesion region. Area of GFAP positive tissue in lesion region corrected for tissue loss. significantly different from OP control and cAMP control.

Area of hPLAP positive cells Area of hPLAP positive cells

25 25

20 20

15 15 GRP control GRP control GRP cAMP GRP cAMP 10 10 P ercen tage (% ) Percentage (%) Percentage 5 5

0 0

A B

Figure 5.21 A,B: Area of hPLAP positive cells in the lesion region. Percentage of tissue in the lesion region occupied by hPLAP positive cells is shown for transverse sections (A) and for horizontal sections (B).

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GRP cell survival and differentiation: GRP cells were present at 84 days after

SCI in all animals that had received transplants and were found to express nestin, GFAP, and CC-1 (Figure 5.22 A-C and 5.23). The quantification method used in this study allowed determination of cell differentiation of a total of 16,046 hPLAP positive cells (8,744 GRP control and 7,302 GRP cAMP); 5,356 were examined for nestin expression, 5,018 for GFAP expression, and 5,672 for CC-1 expression. The proportion of hPLAP positive cells that expressed nestin was 12

± 4% for GRP control and 10 ± 2% for GRP cAMP. The proportion of hPLAP positive cells that expressed GFAP was 60 ± 7% for GRP control and 42 ± 6% for GRP cAMP (p=0.07; F=4.4). The proportion of hPLAP positive cells that expressed CC-1 was 33 ± 5% for GRP control and 41 ± 5% for GRP cAMP.

These results are shown in Figure 5.23.

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A B

Figure 5.22 A-C: Differentiation of hPLAP positive GRP cells. Each confocal image represents one optical slice from a 20 μm section. In A GRP cells are shown that express both hPLAP (red) and nestin (green). In B GRP cells are shown that express both hPLAP (red) and GFAP (green). In C GRP cells are shown that express both hPLAP (red) and CC-1 (green). Cell nuclei are stained with DAPI (blue). Scale bar = 50μm.

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Differentiation of hPLAP cells

50 GRP control 40 GRP cAMP 30 Percentage (%) 20

0 Nestin GFAP CC-1 Cell type

Figure 5.23: Quantification of differentiation of hPLAP positive GRP cells. The percentage of hPLAP positive cells expressing nestin, GFAP, or CC-1 is shown.

Analysis of Lumbosacral Spinal Cord

For 44 animals 20 cells from the DM and DL nuclei in the lumbosacral spinal cord were imaged. The location of the DM and DL nuclei is shown in

Figure 5.24 A and B, respectively. Quantification of the amount of serotonin in close proximity to these motor neurons was significantly less following SCI when compared to the AM control group (p<0.001; F=5.4), however, no differences between groups were found (Figure 5.25). Figure 5.26 shows examples of cells imaged in these nuclei.

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A B Figure 5.24 A,B: Dorsomedial and dorsolateral nuclei in the lumbosacral spinal cord. Both sections are stained for serotonin (green) and cell nuclei (blue) by immunohistochemistry. In A the 2 dorsomedial nuclei are shown left and right from midline just ventral to the central canal. B shows the right dorsolateral nucleus in the ventral horn of the lumbosacral spinal cord.

Serotonergic input to motorneurons

1.8

1.6

1.4 OP control 1.2 GRP control 1 cAMP control GRP cAMP 0.8 AM control 0.6

0.4 Quantification (arbitrary units) 0.2

Figure 5.25: Serotonergic input to motor neurons in the lumbosacral spinal cord. Quantification of serotonin in close proximity to motor neurons shown in arbitrary units for all groups of rats. significantly different from AM control.

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A B

Figure 5.26 A,B: Motor neurons found in the dorsomedial and dorsolateral nuclei. Two representative images of motor neurons located in the dorsomedial or dorsolateral nuclei. Sections are 20 μm thick and stained for serotonin (green), synaptophysin (red), and DNA/RNA (TOTO-3) by immunohistochemistry. The extensive dendritic field with synapses in these nuclei and the serotonergic input (green) to the motor neurons is shown in this figure. Scale bar = 50 μm.

5.4 Discussion

The transplantation technique used in this study did not have a significant

impact on locomotor or autonomic behavioral outcomes; however GRP cells did

alter the cellular composition of the lesion region. The treatment strategy used

here resulted in a significant increase of tissue throughout the lesion region.

Moreover, GRP cells appeared to increase the amount of tissue at the lesion

center. The GRP cells survived, differentiated, and formed extensive transplants

that were well integrated with host tissue in all animals that received a GRP cell

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transplant. Furthermore, in the face of elevated cAMP concentrations these GRP

cells appeared to differentiate more into oligodendrocytes and less into

astrocytes than when cAMP concentrations were not altered. Elevated cAMP

concentrations, however, did reduce the area occupied by GRP cells at the

lesion center and appeared to reduce the volume of GRP cells throughout the

lesion region. The histopathological alterations we found in the lesion region did

not appear to affect serotonergic input to the DM and DL nuclei in the

lumbosacral spinal cord.

Impact parameters produced by the MASCIS-NYU device were similar to

previous reports such as Basso et al., 1996. Our moderate contusion injury

produced a very consistent lesion with the area of remaining tissue at the lesion

center in the OP control group (12.4 ± 2.7%) in the same range as described

previously for this level of injury (9.9 ± 4.8%; Basso et al., 1996). The treatment

interventions studied in this experiment, transplantation of GRP cells and

elevation of cAMP concentrations, did not negatively impact animal health based

on similar weight gain in all experimental groups. However, final locomotor

scores in this study were lower than previously reported for this level of injury at 6 weeks after SCI. (BBB score OP control: 8.2 ± 0.4 vs. 10.3 – 10.6; Basso et al.,

1995 and 1996). Although Basso et al. (1996) examined recovery of males and

females from this level of injury and did not find a significant difference between

the 2 sexes, group numbers were low and weight differences between groups

were not reported. More recently it has been shown that gender differences do

exist during recovery from central nervous system trauma in humans (Stein,

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2001) and rats (O'Connor et al., 2003; Roof and Hall, 2000). Furthermore, a

recent study demonstrated improved recovery from SCI in female rats and mice

when compared to males (Hauben et al., 2002). The exact mechanism for this is

unknown but likely is a result of multiple factors, such as differences in

hormones, immune responses, and size/weight. One group has suggested that

smaller animals recover faster from SCI than larger ones (Bambakidis and Miller,

2004). When comparing our results with other similar studies, one must keep in

mind that we examined relatively heavy male rats unlike most other studies that

have looked at recovery in lighter female rats.

Prior to the start of this experiment, the transplantation technique using

the same FlexiFil syringe was examined in 2 animals with intact spinal cords. No

detrimental effect on hind limb motor function was noticed in these animals when

examined using the BBB locomotor rating scale. However, the impact of this procedure may be larger in injured animals that are just recovering from a

moderate contusion injury. When the BBB locomotor scores are examined

closely one is able to distinguish a short period in which the recovery appears

blunted between 7 and 10 days after SCI. Perhaps the damage from our

transplantation procedure that is negligible in healthy rats was sufficient to negatively affect recovery during a critical phase after SCI in the rats used in this study. From the graph that depicts the movement of animals in their cages, one recognizes a negative impact on activity of the transplantation procedure. In fact it takes approximately 7 days for rats to regain their pre-transplantation surgery level of activity. It has been well established that exercise improves locomotor

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recovery after SCI (Behrman and Harkema, 2000; Engesser-Cesar et al., 2005;

Field-Fote and Tepavac, 2002; Van Meeteren et al., 2003); however it remains to be determined whether a transient decrease of activity during this sub-acute phase after SCI would have a substantial effect on long-term outcome.

Interestingly, a negative effect from the transplantation procedure may

also contribute to what is seen in another study (Mitsui et al., 2005b) in which

rats underwent a similar protocol of SCI followed by transplantation of precursor

cells in 3 sites of the lesion at 9 days following SCI. In this study transplant

injections in the dorsal midline of the spinal cord were performed using a

Hamilton syringe with a tip of 310μm which is significantly larger than what we used (180μm). This study demonstrated a beneficial effect of transplanting a

combination of neuronal precursor (NRP) and GRP cells that resulted in

improvement of the BBB locomotor score from 7.1 (operated control group) to 9.4

(NRP GRP group). The operated control group not only had a low final BBB

score, but also showed a drop in locomotor recovery at 1 week after transplant.

The apparent negative impact of the surgical procedure seen in the operated

control group, which is more dramatic than what we have seen in our study, was not discussed. Mitsui et al. (2005b) attributed the comparatively low BBB score

seen in their operated control group to the modified-moderate contusion injury

they use, in which the impactor rod is left on the spinal cord for an additional 5

sec following a 25 g-cm contusion. However, review of the paper that

demonstrates their modified-moderate contusion model (Mitsui et al., 2005a) indicates that BBB scores are expected to be 3.5 in operated control animals at 8

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weeks after transplant. This latter study (Mitsui et al., 2005a) demonstrated a positive effect of transplanting fibroblasts expressing brain-derived neurotrophic factor (BDNF) and neurotrophin 3 (NT-3) on recovery of locomotor function following SCI in a similar experimental paradigm as described above for their

NRP GRP study. Transplantation of fibroblasts expressing BDNF and NT-3 improved BBB locomotor scores from 3.5 (operated control) to 8.2. From the results of that study no negative impact of the surgical procedure was seen on locomotor recovery.

Another study (Pearse et al., 2004) demonstrated positive effects on locomotor outcome of a combination of transplanting Schwann cells 7 days after a moderate SCI and elevating cAMP concentrations. This group used the same methodology as we did to perform the transplant and elevate cAMP concentrations. Although at 8 weeks after transplant the BBB score of the injured-only group was 10.4 vs. 15.0 in the treated group, at 1 week after transplant the injured-only group had a BBB score of 7 vs 10.5 in the treated group. No BBB scores are shown prior to 1 week after transplant. Unfortunately it is unknown whether pre-existing differences in BBB scores were present.

Furthermore, this does not allow interpretation of acute effects of the

transplantation surgery.

Transplantation of multineurotrophin-expressing GRP cells had positive

effects on locomotor recovery (Cao et al., 2005) as demonstrated by a BBB

score of 15.6 in treated animals vs. 13.1 in operated control animals. The

experimental paradigm was similar to our study and the studies conducted by

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Mitsui et al. (2000a and 2000b), in that transplantation was performed 9 days

after SCI. However, the level of injury (150 kdyn; Infinite Horizon device) was

much less severe and resulted in BBB scores of 10 – 12 prior to transplant and

12.2 – 15.6 (operated control and multitrophin-expressing GRP group,

respectively) at 6 weeks after SCI. This study used a transplantation technique

that involved a total of 4 injections; 2 bilaterally rostral to the lesion region 0.6

mm off midline and 2 bilaterally distal to the lesion region 0.6 mm off midline with

a glass micropipette with an outer diameter of 50-70μm. No negative effects of

the surgical procedure were apparent from the locomotor recovery graphs.

Other studies examining the effects of different glial progenitor cells such

as the human embryonic oligodendrocyte precursor cells (OPCs) (Keirstead et al., 2005) and oligodendrocyte precursors (Bambakidis and Miller, 2004) have demonstrated positive effects on locomotor outcome however these studies were conducted in less severe SCI models (Bambakidis and Miller, 2004; Keirstead et al., 2005). One elegantly performed study in male rats of similar weight as ours, demonstrated a positive effect of transplanting oligodendrocyte-type 2 astrocytes

(O-2A) into the lesion center on locomotor recovery after a 25 g-cm SCI (Lee et

al., 2005). Although a significant difference was found in BBB score at 6 weeks

after transplantation (9.5 in operated control animals vs. 12.3 in treated animals),

few significant effects of transplantation were seen during electrophysiological

assessment of these animals. The O-2A cells used in this study differentiated

into oligodendrocytes that had the ability to promote remyelination and regeneration of axons. Differences between GRP cells and O-2A/OPC cells

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include different chemokine response patterns, different responses to inducers of

differentiation, and different behaviors of the 2 precursor cell populations

following transplantation (Rao et al., 1998). The latter is most striking and reflects the ability of GRP cells to readily generate astrocytes whereas the O-2A/OPC cells only generate oligodendrocytes (Noble et al., 2004). Differences in characteristics of these progenitor cells undoubtedly affect eventual functional outcomes.

Although number of micturitions per 24 hours, micturition duration, and

erectile event duration appeared to have recovered more in the GRP cAMP

treatment groups, none of these outcomes were unequivocally positively affected

by our intervention. We did not find significant beneficial effects of our

transplantation strategy on micturition or sexual outcome measurements. Few

studies have examined effects of cell replacement strategies on micturition

function of which the most interesting are the 2 studies by Mitsui et al. (2005a

and 2005b) of which the locomotor recovery data have been discussed above.

Fibroblasts engineered to secrete BDNF and NT-3 (Mitsui et al., 2005a)

and the combined application of NRP and GRP cells (Mitsui et al., 2005b) appeared to accelerate recovery of micturition from the period of bladder areflexia in rats observed in metabolic cages. Treatment differences were no longer apparent by 4 weeks after transplantation. Both of these treatments also appeared to reduce signs of detrusor sphincter dyssynergia and detrusor hyperreflexia as determined by cystometry and determination of bladder weights.

Considering the enormous difference in BBB locomotor scores between the 2

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groups (3.5 vs 8.2) after transplant of genetically modified fibroblasts, it is not

surprising other behavioral outcomes improved as well. Interestingly,

development of thermal hypersensitivity was not affected by this treatment. The

combined NRP GRP transplant improved BBB scores from 7.1 to 9.4 and also

diminished thermal hypersensitivity. Not only did both of these studies report beneficial effects from their transplantation strategies, investigators also

determined increased serotonergic input to the DL nucleus in the lumbosacral spinal cord; however review of data shown (Mitsui et al., 2005a) questions location of nucleus examined for serotonin. An earlier report (Mitsui et al., 2003) demonstrated beneficial effects from transplanted immortalized neural stem cells on micturition function, however, the experimental paradigm in that study was significantly different and the report did not include locomotor recovery data.

Previously it has been shown that when neurons grown on inhibiting

substrates are exposed to BNDF and NT-3 their regenerating capability is

improved in vitro (Bregman et al., 1998). Subsequent studies showed that this

was mainly due to downstream effects of the cAMP-dependent protein kinase

(PKA) (Cai et al., 1999). Activation of PKA induces transcription of regeneration-

related genes and production of polyamine products. Polyamines have been

shown to be involved in regulation of microtubule assembly and modulation of

expression of cytoskeleton proteins. It was suggested that neurotrophin

treatment resulted in elevated cAMP concentrations that consequently reduced

responsiveness of neurons to inhibitory cues provided by myelin. Preventing

changes induced by the myelin-associated inhibitors occur potentially through

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stabilization and rearrangement of cytoskeletal structures through effects of polyamines (Spencer and Filbin, 2004). Pearse et al. (2004) demonstrated that administration of the PDE-IV inhibitor rolipram and db-cAMP, using the same methodology as we did in our study, prevented the SCI-induced reduction of spinal cord cAMP concentrations. This study demonstrated that the combination of Schwann cell transplants and elevation of cAMP concentrations following SCI resulted in significant improvement of locomotor function and promoted supraspinal and propriospinal axon sparing and myelination. Also, serotonergic fiber growth into and beyond the graft was promoted by this treatment. This study did not quantify extent of transplant grafts within the injury lesion region and whether or not this was affected by cAMP elevation. Anti-inflammatory effects such as prevention of immune cell activation and pro-inflammatory cytokine production have been reported for cAMP. In the study described above, TNF-α production was inhibited but no effect was seen on IL-1β concentrations (Pearse et al., 2004). Another study by this group showed that treatment with rolipram improved functional recovery, promoted axonal growth, and attenuated astrogliosis (Nikulina et al., 2004). Density of sections stained with GFAP was quantitated and in rolipram-treated animals this density was one third less compared to vehicle-treated animals. This is particularly interesting in light of our findings of reduced differentiation of GRP cells into astrocytes. Perhaps reduced activation of endogenous astrocytes and reduced glial scarring is produced through similar mechanisms of cAMP that alter lineage differentiation of GRP cells. In the present study significantly less double labeled hPLAP-GFAP positive

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cells were seen in the animals with elevated cAMP concentrations (GRP cAMP

group). Although more hPLAP-CC-1 positive cells were seen in the GRP cAMP

group this had no beneficial effect on functional outcome. With increased

availability of oligodendrocytes, one would expect to see enhanced re-

myelination and potentially improved functional outcome. However, it is possible

that the total number of oligodendrocytes (hPLAP-positive) was not increased in

our GRP cAMP group since cAMP appeared to have a negative effect on GRP

cell survival.

As far as the author is aware, this is the only study that has quantified extent of transplant within the lesioned spinal cord. Certainly there are no other reports on the effect of cAMP on survival of transplanted cells within an injured spinal cord. Our study suggests that elevation of cAMP is not necessarily beneficial to survival of GRP cells. This is unlikely to be due to the altered immune response that is caused by cAMP elevation, since it has been well established that cAMP has predominantly anti-inflammatory effects that are described above. Macrophages failed to differentiate into activated macrophages when exogenous cAMP was added in an in vitro model (Peters et al., 1990) and phagocytotic function of macrophages is inhibited by cAMP (Aronoff et al., 2005;

Peters et al., 1990). Numerous reports have shown that cAMP is involved in regulation of cell survival, in particular of neoplastic and progenitor cells.

In B-cell chronic lymphocytic leukemia (B-CLL) PDE-IV is the predominant

PDE expressed in the neoplastic cells. Rolipram induces apoptosis in B-CLL

cells through a cAMP dependent and caspase dependent manner. Furthermore,

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rolipram was found to suppress the anti-apoptotic members of the Bcl-2 family

and induced the pro-apoptotic protein Bax. Combining these mechanisms

resulted in a shift of the balance between pro- and anti-apoptotic members of the

Bcl-2 family towards a pro-apoptotic direction (Siegmund et al., 2001). In acute

lymphoblastic leukemia (ALL) cells, elevation of cAMP through rolipram

administration resulted in G1 and G2/M cell cycle arrest and increased apoptosis

(Ogawa, R. et al., 2002a). Chronic lymphocytic leukemia (CLL) cells undergo

apoptosis following administration of rolipram. Contributing mechanisms included

mitochondrial depolarization, release of cytochrome c into the cytosol, and

caspase-9 and -3 activation (Moon and Lerner, 2003). The most recent study

determined that rolipram increased the efficacy of glucocorticoid-mediated apoptosis in B-CLL. PKA was required for this effect and enhanced apoptosis by modulating glucocorticoid receptor signal transduction (Tiwari et al., 2005).

Elevation of cAMP concentration has also been shown to inhibit cell growth and induce apoptosis in other cancer cell lines such as retinoblastoma cells (Fassina

et al., 1997), papilloma cells (Marko et al., 1998), glioma cells (Chen et al., 2002), neuroblastoma cells (Kumar et al., 2004), and esophageal cancer cells (Wang,

H. M. et al., 2005a). In cancer cell lines this effect of PDE-IV inhibition is an obvious area of interest since halting growth of these cells is a much desired therapeutic goal.

In the treatment of bronchial asthma, PDE inhibition is thought to be an

important mechanism of the anti-inflammatory actions of theophylline.

Theophylline and rolipram have been shown to increase eosinophil intracellular

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cAMP concentrations and inhibit eosinophil survival (Momose et al., 1998;

Takeuchi et al., 2002; Wang, W. et al., 2005b). Recently it has been demonstrated that cAMP elevation resulted in significant inhibition of colony growth and induced apoptosis of progenitor cells in asthmatics, but not in normal subjects. These effects were not limited to the eosinophil lineage alone (Wang,

C. H. et al., 2003). A number of possible mechanisms for this exist including internucleosomal DNA cleavage, G1 cell arrest, and effects through CREB gene

regulation. Another potential mechanism for the pro-apoptotic effect of cAMP is given in a recent study of pulmonary hypertension. Pulmonary arterial hypertension is a proliferative vascular disease characterized by aberrant

regulation of smooth muscle cell proliferation and apoptosis in distal pulmonary

arteries. Elevating cAMP concentrations through administration of PDE inhibitors

suppressed proliferation and matrix metalloproteinase activity and promoted

apoptosis in these cells. One of the important mechanisms appeared to be

attenuation of DNA synthesis (Growcott et al., 2006).

In conclusion, this study showed no beneficial effects on functional

outcome measurements of transplanting GRP cells and elevating cAMP

concentrations after SCI. Although elevation of cAMP has been reported to have

beneficial effects in neural regeneration and may have beneficial effects on

lineage differentiation of GRP cells, from our study it appears that cAMP

elevation may reduce survival of GRP cells following transplantation into the

injured spinal cord. Continued research should be undertaken into the effects of these combined treatment strategies for SCI. Transplant strategies appear

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promising; however it seems necessary to combine this with other therapies such as treatments that induce differentiation of these progenitors into a cell population with desired functional characteristics, treatments that affect regeneration, and/or treatments that alter the spinal cord milieu. It is important that investigators critically examine effects of all these treatments, not only on functional outcome measurements but also on histopathological features.

Particularly in combination therapies it is important to also examine interactions of the therapeutics used. Effects of the complimentary treatments such as administered neurotrophins or cAMP elevation may have different effects in normal adult cells vs. in progenitor such as used in current transplantation strategies.

5.5 References

Anderson KD: Targeting recovery: priorities of the spinal cord-injured population. J Neurotrauma 21:1371-1383, 2004.

Aronoff DM, Canetti C, Serezani CH, Luo M, Peters-Golden M: Cutting edge: macrophage inhibition by cyclic AMP (cAMP): differential roles of protein kinase A and exchange protein directly activated by cAMP-1. J Immunol 174:595-599, 2005.

Bambakidis NC, Miller RH: Transplantation of oligodendrocyte precursors and sonic hedgehog results in improved function and white matter sparing in the spinal cords of adult rats after contusion. Spine J 4:16-26, 2004.

Basso DM, Beattie MS, Bresnahan JC: A sensitive and reliable locomotor rating scale for open field testing in rats. J Neurotrauma 12:1-21, 1995.

Basso DM, Beattie MS, Bresnahan JC: Graded histological and locomotor outcomes after spinal cord contusion using the NYU weight-drop device versus transection. Exp Neurol 139:244-256, 1996.

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CHAPTER 6

DISCUSSION

Urination and sexual function are compromised after SCI. The resulting

dysfunction represents a combination of recovery of reflexes and plasticity that occurs after loss of descending input, and that subsequently produces hyperactive reflexes. Treatments aimed at ameliorating secondary injury and enhancing sprouting or regeneration may have positive effects on lumbosacral autonomic function, but more information regarding the normal and pathophysiological eliminative and sexual functions and their responses following reparative therapies is needed to plan treatment strategies. This should be especially true since spinal cord injured people consider recovery of these functions to be of highest priority (Anderson, 2004).

In this sequence of investigations we demonstrate the value of a novel

method to assess autonomic behavior in freely moving rats and apply that

technique to investigate the use of a cell transplantation technique in

experimental SCI. Using a telemetric technique to measure CSP pressure we

demonstrate the ability to assess details of micturition and erectile function in

conscious male rats. Using this technique we found that partial recovery of

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micturition function occurs after mild SCI but that micturition characteristics

remain altered such as demonstrated by a 2-fold increased duration and

increased mean pressures of micturition events 3 weeks after injury. Similar

changes were seen after analysis of number and characteristics of the erectile events. We found a decreased number of both full and partial erectile events following mild SCI and we found significant trends towards increased duration, area under the curve, and maximum pressures of the events as well as trends towards increased peak duration, peak area under the curve, and peak maximum pressures. We suggest this is a result from development of bulbospongiosus muscle spasticity or hyperreflexia after SCI.

Finally, using this technique to assess recovery of micturition and erectile

function after SCI, we evaluated these autonomic outcomes in rats that were

subjected to moderate SCI and received a treatment intervention that included a combination of transplantation of GRP cells and elevation of cAMP concentrations. However, no beneficial effects on functional outcome measurements of this transplantation strategy were observed in this study. We used a more severe level of injury in this latter study since other investigators have shown that less severe injuries have minimal impact on bladder function

(Mitsui et al., 2005; Pikov and Wrathall, 2001). We elected to ensure substantial damage in order to show improvement of autonomic parameters after transplantation of GRP cells on day 9 after SCI. However, in our moderately injured animals we detected more variation within a number of our measurements than was apparent in our mildly injured animals. This may have

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contributed to the lack of finding a significant effect of treatment, although, even

in some of the more robust measurements no significant effect of our therapy

was seen. Since we did observe long-term changes in waveform characteristics

after a mild SCI (Nout, Y. S. et al., 2005b) investigators should consider to

perform future studies of treatment interventions in milder models of SCI.

Furthermore, it appears that beneficial effects from transplantation strategies may be more easily identified following mild SCI. Unless animals recover spontaneously from deficits incurred by mild SCI, less severe models of SCI may prove to be valuable in future investigations of transplant strategies.

Two ways of limiting variability of CSP measurements are increasing the

number of subjects and/or limit the events analyzed. One way to reduce some of

the variability seen in measurements of erectile function would be to only

examine specific erectile events. From our study that examined CSP recording in

uninjured rats, it was apparent that erectile events are context specific as has

been shown before (Schmidt and Schmidt, 2004; Schmidt et al., 1994). For

example, erections that occur during ex-copulatory reflex erection tests are very

different from those that are seen in mating tests. In order to specifically assess

whether the spinal cord is intact, one could limit analysis of erectile events to

those that are non-reflex mediated, in other words, to erectile events that are

produced by supraspinal regulatory areas of the brain. In future research one

could do this by investigating only sleep-related erections. This would require

continuous electroencephalogram recordings of rats in order to only examine

erections that occur during rapid eye movement sleep of the rat.

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Although we did not find behavioral benefits of our treatment we did find that transplanting GRP cells increased the amount of tissue present at the lesion center and we found interesting effects of elevation of cAMP on GRP cell survival and differentiation. From our study it appears that cAMP elevation may reduce survival of GRP cells following transplantation into the injured spinal cord.

Continued research should be undertaken into the effects of these combined treatment strategies for SCI. Current modern imaging and analytical technologies enable scientists to perform detailed histopathological investigations into the roles that these treatments play. Furthermore, continued development of magnetic resonance imaging (MRI) will potentially allow us to examine properties of transplanted cells in vivo.

Up to now, most pathophysiological studies in the field of SCI research have been based on postmortem evaluation of the lesions but recent progresses in the use of MRI provide new tools to examine the rodent central nervous system in vivo, and, eventually monitor progression of lesions. The essential aspect of utilization of MRI in neurotrauma consists in monitoring acute-to- chronic posttraumatic physicochemical changes and correlating them with biochemical and morphological changes ex vivo. We have recently demonstrated the utilization of MRI in a thoracic SCI model in which we compared MRI to histopathological data (Nout, Y.S. et al., 2005a). Moreover, use of MRI may assist in the progress of developing transplant repair strategies for nervous system damage because MRI will permit investigations of these therapies through tracking the fate and time-course of migration of the transplanted cells in

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the host organism in vivo. Following transplanted stem cells by MRI requires a labeling method that will make grafted cells distinguishable from host cells. The contrast produced by the label must be sufficient to permit detection of small cell clusters using high-resolution MRI. Studies have examined the use of gadolinium rhodamine dextran (GRID), which is detectable by both MRI and fluorescence microscopy (Modo et al., 2002), and superparamagnetic iron oxide nanopatricles

(magnetodendimers) (Dunning et al., 2004; Jendelova et al., 2004; Lee et al.,

2004). Although these investigations provide evidence for maintenance of functional properties of graft cells and their migration in spinal cord injury, further development of these techniques remains necessary to improve image quality.

Technical advances to date include imaging techniques such as diffusion tensor

MRI (Lin et al., 2001), use of implantable coils (Bilgen et al., 2001; Silver et al.,

2001), and respiratory gating software (Bonny et al., 2004).

Transplant strategies appear promising, but it is important that investigators critically examine effects of all these treatments, not only on functional outcome measurements but also on histopathological features. There are currently a number of injury models available in rats and mice and laboratories use different behavioral tests and analytical techniques to evaluate their therapeutic strategies. This broad approach to SCI research has the benefit of investigating many different things at the same time; however, the downside is that comparing results between laboratories becomes more difficult. Prior to recommending treatments for clinical trials, it is important that these treatments are evaluated by more than one group of investigators. Rosenzweig and

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McDonald (2004) state that the needs of individuals with SCI dictate that we must improve the comparability of our studies to allow more balanced decisions about the possibilities for translation to human therapies.

In conclusion, the experiments performed here are a start to answer to the request of the spinal cord injured population that more research should be focused on quality of life issues secondary to disruption of autonomic nervous system pathways after injury. The experimental model that we have developed here allows detailed study of micturition and erectile function in rats and should prove valuable in future research focused on these bodily functions after SCI.

None of our behavioral outcomes were affected by the transplantation technique used here, but other valuable information was obtained from this study; the detrimental effect of cAMP on GRP cell survival that we showed here must be further studied. Continued research into the capabilities of precursor transplantation techniques is essential in this road towards finding a cure for SCI.

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APPENDIX

IMAGES FROM SPINAL CORD SECTIONS

A B

Continued

Figure A.1 A-J: Representative images of animals that received GRP cell transplants – transverse sections lesion region. Images A – E are from each of the 5 animals in the GRP control group and images F – J are from each of the 5 animals in the GRP cAMP group. Sections are 20 μm thick and immunohistochemically stained for astrocytes (GFAP = green), GRP cells (hPLAP = red), and cell nuclei (DAPI = blue).

179

Figure A.1 continued

C D

E F

Continued

180

Figure A.1 continued

G H

I J

181

Continued

Figure A.2 A-I: Representative images of animals that received GRP cell transplants – horizontal sections lesion region. Images A – E are from each of the 5 animals in the GRP control group and images F – I are from each of the 4 animals in the GRP cAMP group. Sections are 20 μm thick and immunohistochemically stained for serotonin (green), GRP cells (hPLAP = red), and cell nuclei (DAPI = blue).

182

Figure A.2 continued

Continued

183

Figure A.2 continued

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