ELECTRORETINOGRAPHY: SELECTION of PATIENTS Prior to ERG Or MRI Examination

Scientific Works. Series C. Veterinary Medicine. Vol. LXIII (1)

ISSN 2065-1295; ISSN 2343-9394 (CD-ROM); ISSN 2067-3663 (Online); ISSN-L 2065-1295 Therefore, evaluation of the chromatic PLR in ground electrodes were skin needles electrodes blind patients can suggest a possible diagnosis (Stainless steel subdermal electrode; ELECTRORETINOGRAPHY: SELECTION OF PATIENTS prior to ERG or MRI examination. OcuScience, USA) that were placed AND PERFORMING THE TECHNIQUE Prior to anesthesia, both pupils were dilated by approximately 2 cm caudal to the lateral applying one drop of 1% tropicamide (Tropi- canthus and in the midline on the top of the Adina ARGASEALA, Lia ION, Gina GIRDAN, Ion Alin BIRTOIU, camida 1%; Rompharm, Romania) and phenyl- skull, respectively. Electrodes were kept in a Jacqueline MOCANU, Iuliana IONASCU ephrine 10% (Fenefrin 10%; UnimedPharma, stable position using a piece of tape. All 3 Slovakia) three times, with a 10 minutes electrodes were connected to a preamplifier and Faculty of Veterinary Medicine of Bucharest, 105 Independence Street, District 5, interval between instillation of the drops. the signals were amplified with a bandpass of Bucharest, Romania The patients were fully anesthetized in order to 0.3 to 300 Hz, provided by the HMsERGunit. prevent artifacts due to involuntary muscles Impedance and baseline tests were performed Corresponding author email: [email protected] movement (Ekesten et al., 2013). before ERG recordings. The electrode Dogs were premedicated with dexmedeto- impedance was maintained under 5 kΩ and the Abstract midine (2-10 mcg/kg) (Dexdomitor 0,5 mg/ml; baseline test under 25 microvolts. Orion Pharma, Finland) and butorphanol (0.2- A handheld mini Ganzfeld dome, positioned Electroretinography (ERG) is an objective test that evaluates the retinal function. This paper wants to present to the 0.4 mg/kg) (Butomidor; Richterpharma, approximately 1 cm to the eye, without veterinary Romanian scientific community the importance of using this diagnostic tool for clinicians dealing with blind Austria), delivered intramuscularly. Induction touching the animal, provided light stimuli and patients. Candidates for ERG were selected from ophthalmological patients presented with blindness because it was difficult to establish a definitive diagnosis using only ophthalmological examination. All patients underwent general and maintenance were achieved using propofol background adaptation. anesthesia. ERG was performed using the HMsERG unit, with preprogrammed included protocols. The protocols used initial bolus (1 mg/kg) (Norofol 1%; Maravet, in this study were Short protocol for the cataract surgery candidates and ISCEV protocol for the other patients. ERG Romania) and a constant rate infusion of 0.1- confirmed the results obtained on chromatic pupillary light reflex evaluation. ERG proved useful in the following cases: 0.5 mg/kg/min intravenously. diagnosing retinal inherited disorders, differentiating between retinal and postretinal causes of blindness, evaluation of Cats were premedicated using dexmedeto- retinal function in cataract surgery candidates. ERG objectively assesses the retinal function and should be used on a larger scale by practitioners dealing with blind patients. midine (0.01 mg/kg), ketamine (2 mg/kg) (Anestamine 100 mg/ml; LeVet Pharma, Key words: electroretinography, retina, blindness, cataract. Holland) and butorphanol (0.2 mg/kg) administered intramuscularly. Induction and INTRODUCTION MATERIALS AND METHODS maintenance were achieved using propofol initial bolus (4 mg/kg) and a constant rate The flash electroretinogram (ERG) is an Candidates for ERG were selected from the infusion of 0.6 mg/kg/min. electrodiagnostic test that assesses the function patients presented with blindness in the Local nerve blocks of the auriculopalpebral and of one part of the central nervous system, the Ophthalmology Department of the Faculty of zygomatic nerve were performed on all retina (Gelatt et. al, 2013) and it is described as Veterinary Medicine of Bucharest, during patients, using lidocaine (1-2 mg/kg) (Xilina the electrical response recorded when the retina December 2016 – February 2017. They were 10mg/ml; Zentiva, Romania). is stimulated by flashes of light (Maggs et al., divided into 3 groups, based on clinical Topical anesthesia was induced using 0.4% Figure 1. ERG setup using the HMsERG 2008). findings: partially blind patients, completely oxybuprocaine hydrochloride (Benoxi 0.4%; device. This technique is useful for the early diagnosis blind patients and cataract surgery candidates. UnimedPharma, Slovakia). and prognosis of inherited progressive retinal After clinical assessment, serological and Each patient was placed in sternal recumbency. The HMsERG system software package has atrophy (PRA) or characterizing retinopathy hematological examinations were performed. Eyelids were kept open during the examination several preprogrammed protocols for due to other causes. Ophthalmological examination included with a lid speculum (Barraquer eye speculum; evaluating retinal function. We used the ISCEV ERG is also useful when it is performed before Schirmer tear test, fluorescein test, visual testing Acrivet, Germany) and globes were centered Protocol for patients with partial or complete cataract surgery as well as in diagnosing (menace response, cotton ball test, obstacles with 1-2 conjunctival stay sutures. blindness and the Short Protocol for cataract specific blinding disorders in dogs, such as test), tonometry, pupillary light reflex, chro- ERGs were recorded using the Handheld surgery candidates. sudden acquired retinal degeneration, optic matic pupillary light reflex, ophthalmoscopy. In Multispecies ERG system (HMsERG, Before performing the ISCEV Protocol, the neuritis or cortical blindness (Narfstrom et al., some cases complementary diagnostic tests RetVetCorp, USA) consisting of the unit body, patients were dark adapted for 20 minute. For 2002). have been used, such as ultrasound and MRI. a mini Ganzfeld dome and three electrodes the Short Protocol, the patients were light Although ERG is a widely used technique in The chromatic pupillary light reflex (chromatic (active, reference and ground electrodes). adapted. All lights in the examining room were veterinary ophthalmology, this paper wants to PLR) is a useful method for detecting PLR Retinal signals were recorded using a contact turned off at the ERG test initiation. present to the veterinary Romanian scientific abnormality in sudden acquired retinal lens active electrode (ERG-jet; Fabrinal, According to the HMsERG user’s manual, for community the importance of using this degeneration (SARDS), progressive retinal Switzerland) that was placed on the cornea rods’ function evaluation, responses for atrophy (PRA), or optic pathway disease, thus after applying artificial tear gel (Hypromeloza scotopic ERG procedures were recorded using diagnostic tool for clinicians dealing with blind 2 patients. distinguishing between these 3 diseases that all P 0.5%; UnimedPharma, Slovakia), in order to low intensity stimuli (0.01 cd.s/m of light present with blindness (Terakado et al, 2013; improve conduction. The reference and the stimuli), scotopic standard intensity responses Yeh et al., 2017). 50

Therefore, evaluation of the chromatic PLR in ground electrodes were skin needles electrodes blind patients can suggest a possible diagnosis (Stainless steel subdermal electrode; ELECTRORETINOGRAPHY: SELECTION OF PATIENTS prior to ERG or MRI examination. OcuScience, USA) that were placed AND PERFORMING THE TECHNIQUE Prior to anesthesia, both pupils were dilated by approximately 2 cm caudal to the lateral applying one drop of 1% tropicamide (Tropi- canthus and in the midline on the top of the Adina ARGASEALA, Lia ION, Gina GIRDAN, Ion Alin BIRTOIU, camida 1%; Rompharm, Romania) and phenyl- skull, respectively. Electrodes were kept in a Jacqueline MOCANU, Iuliana IONASCU ephrine 10% (Fenefrin 10%; UnimedPharma, stable position using a piece of tape. All 3 Slovakia) three times, with a 10 minutes electrodes were connected to a preamplifier and Faculty of Veterinary Medicine of Bucharest, 105 Independence Street, District 5, interval between instillation of the drops. the signals were amplified with a bandpass of Bucharest, Romania The patients were fully anesthetized in order to 0.3 to 300 Hz, provided by the HMsERGunit. prevent artifacts due to involuntary muscles Impedance and baseline tests were performed Corresponding author email: [email protected] movement (Ekesten et al., 2013). before ERG recordings. The electrode Dogs were premedicated with dexmedeto- impedance was maintained under 5 kΩ and the Abstract midine (2-10 mcg/kg) (Dexdomitor 0,5 mg/ml; baseline test under 25 microvolts. Orion Pharma, Finland) and butorphanol (0.2- A handheld mini Ganzfeld dome, positioned Electroretinography (ERG) is an objective test that evaluates the retinal function. This paper wants to present to the 0.4 mg/kg) (Butomidor; Richterpharma, approximately 1 cm to the eye, without veterinary Romanian scientific community the importance of using this diagnostic tool for clinicians dealing with blind Austria), delivered intramuscularly. Induction touching the animal, provided light stimuli and patients. Candidates for ERG were selected from ophthalmological patients presented with blindness because it was difficult to establish a definitive diagnosis using only ophthalmological examination. All patients underwent general and maintenance were achieved using propofol background adaptation. anesthesia. ERG was performed using the HMsERG unit, with preprogrammed included protocols. The protocols used initial bolus (1 mg/kg) (Norofol 1%; Maravet, in this study were Short protocol for the cataract surgery candidates and ISCEV protocol for the other patients. ERG Romania) and a constant rate infusion of 0.1- confirmed the results obtained on chromatic pupillary light reflex evaluation. ERG proved useful in the following cases: 0.5 mg/kg/min intravenously. diagnosing retinal inherited disorders, differentiating between retinal and postretinal causes of blindness, evaluation of Cats were premedicated using dexmedeto- retinal function in cataract surgery candidates. ERG objectively assesses the retinal function and should be used on a larger scale by practitioners dealing with blind patients. midine (0.01 mg/kg), ketamine (2 mg/kg) (Anestamine 100 mg/ml; LeVet Pharma, Key words: electroretinography, retina, blindness, cataract. Holland) and butorphanol (0.2 mg/kg) administered intramuscularly. Induction and INTRODUCTION MATERIALS AND METHODS maintenance were achieved using propofol initial bolus (4 mg/kg) and a constant rate The flash electroretinogram (ERG) is an Candidates for ERG were selected from the infusion of 0.6 mg/kg/min. electrodiagnostic test that assesses the function patients presented with blindness in the Local nerve blocks of the auriculopalpebral and of one part of the central nervous system, the Ophthalmology Department of the Faculty of zygomatic nerve were performed on all retina (Gelatt et. al, 2013) and it is described as Veterinary Medicine of Bucharest, during patients, using lidocaine (1-2 mg/kg) (Xilina the electrical response recorded when the retina December 2016 – February 2017. They were 10mg/ml; Zentiva, Romania). is stimulated by flashes of light (Maggs et al., divided into 3 groups, based on clinical Topical anesthesia was induced using 0.4% Figure 1. ERG setup using the HMsERG 2008). findings: partially blind patients, completely oxybuprocaine hydrochloride (Benoxi 0.4%; device. This technique is useful for the early diagnosis blind patients and cataract surgery candidates. UnimedPharma, Slovakia). and prognosis of inherited progressive retinal After clinical assessment, serological and Each patient was placed in sternal recumbency. The HMsERG system software package has atrophy (PRA) or characterizing retinopathy hematological examinations were performed. Eyelids were kept open during the examination several preprogrammed protocols for due to other causes. Ophthalmological examination included with a lid speculum (Barraquer eye speculum; evaluating retinal function. We used the ISCEV ERG is also useful when it is performed before Schirmer tear test, fluorescein test, visual testing Acrivet, Germany) and globes were centered Protocol for patients with partial or complete cataract surgery as well as in diagnosing (menace response, cotton ball test, obstacles with 1-2 conjunctival stay sutures. blindness and the Short Protocol for cataract specific blinding disorders in dogs, such as test), tonometry, pupillary light reflex, chro- ERGs were recorded using the Handheld surgery candidates. sudden acquired retinal degeneration, optic matic pupillary light reflex, ophthalmoscopy. In Multispecies ERG system (HMsERG, Before performing the ISCEV Protocol, the neuritis or cortical blindness (Narfstrom et al., some cases complementary diagnostic tests RetVetCorp, USA) consisting of the unit body, patients were dark adapted for 20 minute. For 2002). have been used, such as ultrasound and MRI. a mini Ganzfeld dome and three electrodes the Short Protocol, the patients were light Although ERG is a widely used technique in The chromatic pupillary light reflex (chromatic (active, reference and ground electrodes). adapted. All lights in the examining room were veterinary ophthalmology, this paper wants to PLR) is a useful method for detecting PLR Retinal signals were recorded using a contact turned off at the ERG test initiation. present to the veterinary Romanian scientific abnormality in sudden acquired retinal lens active electrode (ERG-jet; Fabrinal, According to the HMsERG user’s manual, for community the importance of using this degeneration (SARDS), progressive retinal Switzerland) that was placed on the cornea rods’ function evaluation, responses for atrophy (PRA), or optic pathway disease, thus after applying artificial tear gel (Hypromeloza scotopic ERG procedures were recorded using diagnostic tool for clinicians dealing with blind 2 patients. distinguishing between these 3 diseases that all P 0.5%; UnimedPharma, Slovakia), in order to low intensity stimuli (0.01 cd.s/m of light present with blindness (Terakado et al, 2013; improve conduction. The reference and the stimuli), scotopic standard intensity responses Yeh et al., 2017). 51

(Std R&C) using 3 cd.s/m2 of light intensity for The a-wave amplitude was measured from the All results were compared with the normal stimulus, scotopic higher intensity responses baseline to the a-wave trough, the b-wave ERG recorded in our clinic for the same species (Hi-Int R&C) using 10 cd.s/m2 of light inten- amplitude was measured from the a-wave and age group and also compared to specialty sity to stimulate both rods and cones. For trough to the b-wave peak. A- and b-wave literature data (Figure 2). cones’ function evaluation, responses for implicit times are calculated from the light photopic ERG procedures were recorded using stimulus onset to the a-wave trough and b-wave light stimuli of 3 cd.s/m2, scotopic higher peak, respectively. Amplitude of the cone intensity responses (HiCones) using 10 cd.s/m2 flicker response represents the average of the of light intensity, flicker responses using 3 amplitudes from the trough to the positive peak cd.s/m2 of light intensity for standard flicker in at least 3 responses in the train. Implicit time 2 (Std. Flicker) and 10 cd.s/m of light intensity of the cone flicker response is calculated as the Figure 5. Fundus examination in a blind 6 months old for higher intensity flicker (Hi-Int. Flicker). average of the times from the light onset to the Husky revealed vasculature attenuation. ERG data for each patient were recorded positive peak in at least 3 responses in the train. bilaterally. ERG recordings were analyzed by (Ekesten et al, 2012). For the scotopic low Hi-int R&C 10 cd.s/m2 measuring the amplitude and implicit time for intensity responses and photopic 30 Hz flicker 100 each a- and b-wave ERG component, as well as responses, only the b-wave amplitude and the b/a ratio. implicit time were measured. 50

Figure 3. The normal fundus of a cat (DSH, 4 years old) 0 b-wave -50 0 50 100 150 200 Hi-int R&C 10cd.s/m2 -50 Normal ERG Patient's ERG

300 ()microvolts) Amplitude -100 Implicite time (msec) 200 Figure 6. The electroretinogram of the Husky confirmed 100 absence of retinal function, consistent with early onset 0 retinal degeneration (PRA) -50 0 50 100 150 200 -100 -200 Amplitude (microvolts) (microvolts) Amplitude a-wave Implicite time (msec)

Figure 4. Normal ERG of the same cat

For the selected ophthalmological patients included in this paper, except for the cataract Figure 2. A- and b-wave parameters in a normal ERG, DSH, 4 years old. The vertical green line represents the stimulus onset surgery candidates, it was difficult to establish a definitive diagnosis using only

RESULTS AND DISCUSSIONS Local anesthetic blocks provided good ophthalmological examination. In patients which were presented with partial or Figure 7. Normal fundus appearance anesthesia, no blinking was recorded, thus of a blind 7 years old French Bulldog Although different from other anesthetic reducing the artifacts to minimum. complete blindness and with ophthalmoscopic protocols usually used for ERG recordings, the The HMsERG device has built in protocols modifications, ERG was used to confirm the protocol that we used assured appropriate based on the International Society for Clinical absence of retinal function, establishing the anesthesia for ERG examination. None of the Electrophysiology of Vision (ISCEV) diagnosis of early or late onset retinal patients were intubated during ERG evaluation. guidelines. We chose the Short Protocol for the degeneration (Figure 5 and Figure 6). Because atipamezole can be used to counter the cataract patients, due to the fact that we only Two of the patients presented with acute effect of dexmedetomidine if needed and needed confirmation whether the retina is blindness, complete mydriasis, unremarkable because propofol is a short action drug, we functional or not and because of the shorter fundus examination and unresponsive pupils to believe that this protocol is safe. Furthermore, time necessary to perform it. The ISCEV the chromatic PLR suggesting optic neuritis. if after ERG recordings, further cataract protocol was used in the cases where we For these patients, ERG recordings proved to be an invaluable tool, diagnosing decreased surgery or MRI examination is needed, the needed a more complex investigation of the anesthesia can be maintained by intubating the retina, separating the rods’ responses from the retinal function, suggesting combined Figure 8. The electroretinogram of the French Bulldog patients. cones’ responses. autoimmune retinal and optic nerve disease (Figure 7 and Figure 8). showed decreased retinal function, consistent with an autoimmune mediated retinitis 52

Figure 3. The normal fundus of a cat (DSH, 4 years old) 0 b-wave -50 0 50 100 150 200 Hi-int R&C 10cd.s/m2 -50 Normal ERG Patient's ERG

Figure 4. Normal ERG of the same cat

One patient presented with partial blindness. CONCLUSIONS Visual tests were negative in the affected eye, pupils were mydriatic in ambiental light and the Electroretinography objectively evaluates the BACTERIAL BIOFILMS AS WOUND HEALING DRESSING – A REVIEW chromatic PLR was normal in both eyes. An retinal function. 1 1 2* 1 Ioana M. BODEA , Aurel MUSTE , Giorgiana M. CĂTUNESCU , Cosmin MUREŞAN ERG was performed and it showed normal Candidates presented with blindness were retinal function. The MRI examination diagnosed with retinal inherited disorders, 1University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca, confirmed a postretinal cause of blindness, differentiating between retinal and postretinal Faculty of Veterinary Medicine, 3-5 Mănăştur Street, 400372, Romania tumor of the coroidal plexus and ventricular causes of blindness. 2University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca, hydrocephalus. Evaluation of retinal function of cataract Faculty of Agriculture, 3-5 Mănăştur Street, 400372, Romania Five patients that were candidates for cataract surgery candidates proved very useful in surgery, were first examined by ERG. A good deciding whether the surgery is recommended. Corresponding author email: [email protected] candidate for surgery has normal retinal ERG should be used on a larger scale by function (established by chromatic PLR and practitioners dealing with blind patients. Abstract ERG) and the lens in a normal, anatomical position. The retinal function responses that Tissue engineering and regenerative medicine promote skin regeneration through biomaterials that are easy to provide. REFERENCES Lately, many studies showed that bacterial biofilms can ensure the necessary conditions for proper healing. Several were recorded helped us in selecting the better bacteria (Acetobacter spp., Lactobacillus spp., Azotobacter spp.) produce extracellular polysaccharides (cellulose, candidates for surgery, as the ones with low a- kefiran, alginate) organized in biofilms with different chemical structures. All have properties that grant medical and b-wave amplitudes and implicit time were Ekesten B., Komaromy A.M., Ofri, R., Petersen-Jones application: cartilage and bone repair, nerve surgery and arterial stent coating. Bacterial cellulose, alginate and S.M., Narfstrom K., 2013. Guidelines for clinical kefiran biofilms seem to have the qualities needed as wound healing dressings, but their characteristics and availability excluded. One of these patients had normal, electroretinography in the dog: 2012 update. Doc promt and complete chromatic PLR, but due to vary widely. The aim of this study was to summarize the current state of art on bacterial biofilms to discriminate among Ophthalmol 127:79–87. their specific properties and application in wound healing management. The comparison was focused on obtaining the low retinal function recorded by the ERG, it Gelatt K.N., Gilger B.C., Kern T.J., 2013. Veterinary th techniques, physiochemical characteristics, advantages and disadvantages of use. Cellulose, alginate and kefiran was excluded from cataract surgery (Figure 9). Ophthalmology, 5 edition, Vol. 1, Wiley-Blackwell, showed good results in wound healing processes, but it seems that cellulose and kefiran are the most used. Biocellulose UK, 684. can be obtained in multiple ways (such as stationary or agitated culture) thus the protocol varies depending on Maggs, D.J., Miller, P.E, Ofri, R., 2008. Slatter’s th available laboratory equipment. Both cellulose and kefiran have high biocompatibility, kefiran presents antimicrobial Fundamentals of Veterinary Ophthalmology, 4 activity as well, while cellulose can incorporate drugs. Alginate has all the properties of a wound dressing material, but Edition, Elsevier, Missouri, 293-294. it is difficult to obtain. In conclusion, bacterial cellulose seems to be the most suitable for local covering of wounds. It is Narfstrom, K., 2002.Electroretinography in veterinary studied extensively on laboratory animals and it is currently used in human medicine. However, there seems to be a medicine –easy or accurate? Vet Ophthalmol, 5, 249- lack of case studies on wound management of small animals, mainly cats and dogs. 251. Terakado K., Yogo T., Nezu Y., Harada Y., Hara Y., Key words: alginate, bacterial biofilms, cellulose, kefiran, wound healing, cellulose. Tagawa M., 2013. Efficacy of the Use of a Colorimetric Pupil Light Reflex Device in the Diagnosis of Fundus Disease or Optic Pathway INTRODUCTION they can contain drugs and bioactive substances Disease in Dogs. J. Vet. Med. Sci. 75(11): 1491– that can accelerate wound healing process 1495. The main function of the skin is to protect the (Sulaeva et al., 2015). Yeh C.Y.,Koehl C.L., Harman C.D., Iwabe S., Guzman body against the environment and major Thus, the dressing of choice must ensure the J., Petersen-Jones S.M., Kardon R.H., Komaromy disorders (chronic infection or necrosis).Wound necessary conditions: a moist and clean A.M., 2017.Assessment of Rod, Cone, and Intrinsically PhotosensitiveRetinal Ganglion Cell healing is linked to growth and regeneration. environment, blood and excess exudates Contributions to the CanineChromatic Pupillary Tissue engineering and regenerative medicine absorption, infection prevention, optimal Figure 9. Differences between ERGs of two cataract Response. Invest Ophthalmol Vis Sci., 58:65–78. employ materials that support and accelerate temperature, non-adhesive and rare changes surgery candidates. If the electric response of the retina healing (Nasrabadi and Ebrahimi, 2011). Thus, (Boateng et al., 2008). Materials must be safe, is good, the patient is then operated (blue line). If the electric response of the retina is low, the surgery is not wound therapy remains a clinical problem and biocompatible, biodegradable and non-toxic. A recommended (red line) a proper, efficient management is required. The variety of materials meet these qualities, such proper treatment needs to promote rapid as chitosan, collagen, gellan gum and bacterial healing and generate functional tissues biofilms (Mokhtarzadeh et al., 2016). (Sulaeva et al., 2015). New approaches are Biofilms are bacterial-synthesised exopoly- being developed for acute and chronic wound saccharide organised into long polysaccharidic that avoid complications. Wound dressings and chains of sugars (glucose or galactose) or sugar medication form an important segment of the derivates arranged in branches (Chawla, 2009). global pharmaceutical market (Patel et al., Their formation is an essential stage in the 2012).The global market attempts to offer a survival of bacteria (Sabra et al., 2001). variety of wound dressings for proper wound Biocellulose is a non-toxic, hypoallergenic, management based on different types of non-biodegradable material, with a unique materials – natural or synthetic. Applicable in nanofiber and porous structure. These different forms – films, hydrocolloids and gels, properties make it a perfect wound dressing 54