Diabetes Publish Ahead of Print, published online March 24, 2008 Abnormal Muscle Spindle Innervation and Large-Fiber Neuropathy in Diabetic Mice Karra A. Muller, B.S.1, Janelle M. Ryals, B.S.1, Eva L. Feldman, M.D., Ph.D.2 and Douglas E. Wright, Ph.D.1 1Department of Anatomy and Cell Biology University of Kansas Medical Center Kansas City, KS 66160 2Department of Neurology University of Michigan Ann Arbor, Michigan 48109 Running Title: Spindle Innervation in Diabetic Mice Corresponding Author: Douglas Wright, Ph.D. Department of Anatomy and Cell Biology University of Kansas Medical Center Kansas City, KS 66160 [email protected] Received for publication 7 January 2008 and accepted in revised form 21 March 2008. Copyright American Diabetes Association, Inc., 2008 Spindle Innervation in Diabetic Mice ABSTRACT Objective. Large-fiber DPN leads to balance and gait abnormalities, placing patients at risk for falls. Large sensory axons innervating muscle spindles provide feedback for balance and gait and, when damaged, can cause altered sensorimotor function. This study aimed to determine whether symptoms of large-fiber DPN in type 1 and 2 diabetic mouse models are related to alterations in muscle spindle innervation. In addition, diabetic mice were treated with insulin to assess whether sensorimotor and spindle deficits were reversible. Research Design and Methods. Behavioral assessments were performed in untreated and treated streptozotocin (STZ)-injected C57BL/6 mice to quantitate diabetes-induced deficits in balance and gait. Quantification of Ia axon innervation of spindles was carried out using immunohistochemistry and confocal microscopy on STZ-injected C57BL/6 and db/db mice. Results. STZ-injected C57BL/6 mice displayed significant and progressive sensorimotor dysfunction. Analysis of Ia innervation patterns of diabetic C57BL/6 spindles revealed a range of abnormalities suggestive of Ia axon degeneration and/or regeneration. The multiple abnormal Ia fiber morphologies resulted in substantial variability in axonal width and inter-rotational distance (IRD). Likewise, db/db mice displayed significant variability in their IRDs compared to db+ mice, suggesting that damage to Ia axons occurs in both type 1 and 2 models. Insulin treatment improved behavioral deficits and restored Ia fiber innervation comparable to nondiabetic mice. Conclusions. Similar to small fibers, Ia axons are vulnerable to diabetes and their damage may contribute to balance and gait deficits. In addition, these studies provide a novel method to assay therapeutic interventions designed for diabetes-induced large-fiber dysfunction. 2 Spindle Innervation in Diabetic Mice stimates from the Centers for Disease spindle that could contribute to increased falls Control and Prevention suggest that and ataxia seen in the elderly (13,14). In E 60-70% of diabetic patients develop rodents, Egr3 null mutant mice have muscle neuropathy. In addition, diabetes is the spindle degeneration postnatally, resulting in leading cause of neuropathy in the United an ataxic gait (15). Therefore, it is plausible States and Western countries (1). Diabetic that symptoms of large-fiber DPN could sensorimotor polyneuropathy (DPN) affects result from damage to muscle spindle large both large and small sensory afferent nerve afferent fibers. fibers. The majority of research focuses on Here, we examined the streptozotocin small-fiber neuropathy leading to increased or (STZ)-induced type 1 and leptin receptor-null decreased pain and temperature sensations (2- mutant type 2 diabetic mouse models for 5). Consequently, there is a shortage of evidence of large-fiber DPN. Our results animal model research exploring large-fiber suggest the presence of STZ-induced large- DPN, which can cause deficits in lower limb fiber DPN using a behavioral test of proprioception, decreased tactile sensitivity sensorimotor function, and muscle spindle and vibration sense, and incoordination due to quantification reveals Ia afferent morphologic balance abnormalities (1,6). changes in the width of the axon and the inter- The sensorimotor deficits resulting from rotational distance (IRD) of annulospiral large-fiber DPN, while sometimes subtle in endings. The leptin receptor-null mutant mice nature, can lead to significant impairment. also displayed aberrant muscle spindle Numerous human studies report that patients morphology similar to type 1 diabetic mice. with DPN are at increased risk for falls due to Importantly, insulin treatment to STZ-induced decreased postural control, altered gait and diabetic mice improved behavioral balance, and increased body sway (7-9). The performance and normalized Ia axon underlying neurologic mechanisms involved morphology in muscle spindles. These novel in large-fiber DPN remain poorly understood. results provide evidence that disrupted muscle It has been hypothesized that large-fiber DPN spindle innervation in both type 1 and 2 instability could be caused by altered diabetic models may be involved, at least in sensorimotor function, specifically damaged part, in the sensorimotor deficits displayed in group Ia and II sensory afferent fibers in STZ-induced DPN. muscle spindles (10). Muscle spindles found within skeletal RESEARCH DESIGN AND METHODS muscle are rapidly adapting sensorimotor Male C57BL/6 mice were purchased at 7 receptors. Spindles in mice are surrounded by weeks of age from Charles River extrafusal muscle fibers and consist of bag (Wilmington, MA) and housed 2 mice/cage and chain intrafusal fibers. Three subtypes of on a 12:12-hour light/dark cycle in the animal nerves innervate the intrafusal fibers: group Ia facility at the University of Kansas Medical and II large sensory axons and gamma motor Center under pathogen-free conditions. Male axons. Muscle spindles are involved in many db/db mice (strain name: BKS.Cg- sensorimotor behaviors such as the regulation m+/+Leprdb/J, stock number: 000642, of proprioception, balance, gait, and the background strain C57BLKS/J) were postural response (11,12), and spindle damage purchased from Jackson Laboratories (Bar can lead to deficits such as incoordination. Harbor, ME), and were housed at the Multiple studies in humans reported University of Michigan under pathogen free morphologic changes to the aging muscle conditions on a 12:12-hour light/dark cycle. A 3 Spindle Innervation in Diabetic Mice breeding colony was established, and mice db/db mice. Diabetes was confirmed in db/db were genotyped after birth. All animals had mice at 8 weeks of age. Immunohistochemical free access to water and mouse chow analysis and quantification of muscle spindles (C57BL/6 mice: Harlan Teklad 8604, 4% kcal were performed on medial gastrocnemius derived from fat; db/db and db+ mice: muscles from 24 week old homozygous LabDiet 5001, 12% kcal derived from fat), (db/db; n=3) and heterozygous (db+; n=3) and use was in accordance with NIH leptin receptor-null mutant mice. guidelines and approved by the University of Beam-walk. Mice were trained during week 2 Kansas Medical Center Animal Care and Use and 3 post-STZ to traverse a 1 m long wooden Committee or the University of Michigan beam with a diameter of 1.2 cm (adapted from Committee on the Care and Use of Animals, 17,18). The animals were recorded for 3 respectively. trials/session on weeks 3, 5 and 10 post-STZ STZ-injected C57BL/6 mice. Diabetes was injection. The behavior task was recorded induced in 8 week-old C57BL/6 mice (n=10) using a digital video camera. A footslip was by a single intraperitoneal injection of STZ counted if either the left or right hindpaw (Sigma, St. Louis, MO) at 180 mg/kg body slipped off the beam. The number of weight (16). Nondiabetic mice (n=8) were footslips/mouse was averaged, and the data injected with 400 µl of the vehicle buffer. was analyzed using a two-way RM ANOVA Hyperglycemia and diabetes was defined as a with Fisher’s PLSD post hoc test. blood glucose level greater than 16mM (~288 Footprinting. Footprinting was used as mg/dl). Weight and tail vein blood glucose previously described in Taylor et al. (18). The levels were measured using glucose rear paws of C57BL/6 mice were inked and diagnostic reagents (Sigma) and analyzed mice walked along a 6 cm X 70 cm track using a two-way repeated measures analysis lined with paper at 10 weeks post-STZ. For of variance (RM ANOVA) with Fisher’s each tracking, 3-5 prints were analyzed for protected least significant difference (PLSD) step length, toe spread (distance between toes post hoc test. 1 through 5), intermediate toe spread (inter- Insulin administration. In a separate group of toe spread; distance between toes 2 through animals, insulin pellets were administered to 5 4), and print length. Means were calculated STZ-induced C57BL/6 diabetic mice, while 6 for each animal and analyzed using unpaired diabetic and 6 nondiabetic mice received t-tests. sham palmitic acid pellets. Insulin Grid-walk. The grid-walk apparatus consisted replacement therapy began 6 weeks post-STZ of an elevated 1.1 cm wire grid that was 20 injection via LinBit sustained release insulin cm X 35 cm, adapted from Onyszchuk et al. pellets (13 +/- 2 mg each; 0.1 U/24 (19). Animals were recorded with a digital hours/pellet; LinShin Canada, Inc., video camera for 5 minutes while walking on Scarborough, Ontario, Canada) implanted the grid at 10 weeks post-STZ injection. subcutaneously in the dorsal skin (2 pellets Hindpaw steps and the number of hindpaw for the first 20g body weight and an foot faults, or slips were counted. The percent additional pellet for every additional 5g body slips were calculated for each animal weight). All pellets remained in the mice for 4 (hindpaw slips/total hindpaw steps x 100), weeks, and an additional insulin pellet was and analyzed using unpaired t-tests. added if blood glucose levels failed to drop Rotorod. The rotorod (AccuRotor Rota Rod, below 16mM after one week. Weight and AccuScan Instruments Inc.; Columbus, OH) blood glucose levels were measured and was used as described in Taylor et al.