Luc Poncelet

Immunolocalization of the calcium binding S100A1, S100A5 and S100A6 proteins in the dog cochlea during postnatal development.

The immunolocalization of three members of the S100 calcium-binding protein family was investigated in the dog cochlea during normal postnatal development. Sections of decalcified and paraffin-embedded cochleae from 16 beagle puppies aged from birth to 3 months were treated with polyclonal antisera raised against the human recombinant S100A1, S100A5, and S100A6 proteins. At birth, in the dog cochlea, S100A1 was expressed in the immature Deiters cells, and slightly in the pillar cells. From the second week, S100A1 was detected in the supporting structures of the organ of Corti, i.e. the Deiters, the pillar, the border, and the Hensens cells, and in the reticular membrane. From birth onwards, S100A5 remained a neuronal-specific protein, only located in a subpopulation of neurons in the spiral ganglion. S100A6 was not expressed at birth. From the second week of life, the Schwann cells and nerve sheaths in the modiolus, in the spiral ganglion, and running in the direction of the organ of Corti exhibited S100A6-labeling. From the 12th postnatal day, some scattered intermediate cells started to express S100A6 protein in the stria vascularis. The number of labeled intermediate cells increased during the third week. At adult stage, the intermediate cells were S100A6-stained with cytoplasmic labeling throughout the stria vascularis from the base to the apex of the cochlea. None of the other cochlear structures expressed the S100 proteins under study during the postnatal development of the dog cochlea. The S100A1, S100A5, S100A6 immunostaining was limited to specific cell types in dog cochlea. These S100 proteins were useful markers in the study of supporting cells, neurons, nerve fibers sheaths and stria vascularis (S100A6) during the normal postnatal development of the dog cochlea.

Inherited polyneuropathy in Leonberger dogs: a mixed or intermediate form of Charcot-Marie-Tooth disease?

A spontaneous distal, symmetrical polyneuropathy in related Leonberger dogs with onset between 1 to 9 years of age was characterized clinically, electrophysiologically, histologically, and morphometrically. Exercise intolerance and weakness was associated with a high‐steppage pelvic‐limb gait, a loss or change in the pitch of the bark, and dyspnea. Neurological examination revealed marked atrophy of the distal limb muscles, depressed spinal and cranial nerve reflexes, and weak or absent movement of the laryngeal and pharyngeal muscles. Electrophysiological evaluation was consistent with denervation and was characterized by loss or marked attenuation of compound muscle action potentials and slowed motor nerve conduction velocity. Muscle biopsy specimens showed neurogenic atrophy. Chronic nerve fiber loss associated with decreased myelinated fiber density and a shift of the axonal size–frequency distribution toward smaller fibers was the predominant finding in peripheral nerve specimens. Pedigree analysis of a large multigenerational family, including nine sibships with at least one affected individual, suggested X‐linked inheritance. Mutational and linkage analysis of this family may aid in identification of the chromosomal loci and gene responsible for this inherited axonal neuropathy. Further characterization of this inherited axonal neuropathy may establish the Leonberger dog as a spontaneous animal model of inherited axonal neuropathy and possibly lead to the discovery of a new gene or genes associated with axonal variants. Muscle Nerve 27: 471–477, 2003

An original inner ear neuroepithelial degeneration in a deaf Rottweiler puppy.

Histopathological investigation was conducted on both inner ears from a 4.5-month-old Rottweiler puppy with electrophysiologically confirmed bilateral deafness. The lesions were restricted to the organ of Corti and spiral ganglion that both displayed severe degenerative changes. The outer hair cells were less affected than the inner hair cells. The number of spiral ganglion neurons was reduced, and remaining neurons were altered. The basal and middle cochlear turns were more affected than the apical one. The vestibules were normal. Immunostaining with calbindin, calretinin, S100A1 and S100A6 polyclonal antisera was helpful in identifying different cell-types in the degenerated cochlea. The early and severe spiral ganglion cell degeneration is an uncommon finding no matter the species. Such lesions bear significance within the frame of cochlear implants technology for deaf infants.

Clinical and electrophysiological characterization of myokymia and neuromyotonia in Jack Russell Terriers

BACKGROUND Generalized myokymia and neuromyotonia (M/NM) in Jack Russell Terriers (JRTs) is related to peripheral nerve hyperexcitability syndrome in humans, a symptom complex resulting from diverse etiologies. OBJECTIVE Clinical and electrodiagnostic evaluation is used to narrow the list of possible etiological diagnoses in JRTs with M/NM. ANIMALS Nine healthy JRTs and 8 affected JRTs. METHODS A prospective study was conducted comparing clinical and electrophysiological characteristics in 8 JRTs affected by M/NM with 9 healthy JRT controls. RESULTS All affected dogs except 1 had clinical signs typical of hereditary ataxia (HA). In 6 dogs, neuromyotonic discharges were recorded during electromyogram. Motor nerve conduction studies showed an axonal neuropathy in only 1 affected dog. Compared with controls, brainstem auditory-evoked potentials (BAEP) showed prolonged latencies (P<.05) accompanied by the disappearance of wave components in 3 dogs. Onset latencies of tibial sensory-evoked potentials (SEP) recorded at the lumbar intervertebral level were delayed in the affected group (P<.001). The BAEP and SEP results of the only neuromyotonic dog without ataxia were normal. CONCLUSIONS AND CLINICAL IMPORTANCE The BAEP and spinal SEP abnormalities observed in JRTs with M/NM were associated with the presence of HA. Therefore, these electrophysiological findings presumably arise from the neurodegenerative changes characterizing HA and do not directly elucidate the pathogenesis of M/NM. An underlying neuronal ion channel dysfunction is thought to be the cause of M/NM in JRTs.

Immunolocalization of calbindin D28k and calretinin in the dog cochlea during postnatal development

The calbindin (CB) and the calretinin (CR) immunoreactivities were studied in the dog cochlea during its postnatal maturation from birth to the 33rd postnatal day. At birth, CB was expressed in the Köllikers organ, in the immature inner (IHC) and outer hair cells (OHC), in neurons of the spiral ganglion, and in nerve fibers running in the basilar membrane of the apical turn. During the cochlear maturation, non-sensorineuronal structures, such as the Köllikers organ, the rods of Corti, and the inner sulcus cells, displayed a transient CB-staining. In the adult-like dog cochlea, CB was found in the cytoplasm, the cuticular plate, and the stereocilia of the IHC and OHC. All the neurons of the spiral ganglion and some nerves fibers in the modulius were CB-positive. At birth, CR exhibited a neuronal distribution: about 75% of the spiral ganglion neurons, some nerve fibers in the modulius and nerve fibers running in the basilar membrane were CR-labeled. During the postnatal maturation, a CR-immunostaining appeared around the IHC body and CR was expressed transiently in the OHC. In the adult-like dog cochlea, a CR-positive network surrounded the unlabeled IHC. The neuronal CR-labeling remained unchanged from birth.

Naturally Occurring Parvovirus-associated Feline Hypogranular Cerebellar Hypoplasia—A Comparison to Experimentally-induced Lesions Using Immunohistology:

Three cases of feline cerebellar hypoplasia are presented. At the time of examination, the ages of the cats ranged from 2 months to 1 year. Necropsy revealed cerebellar and pons hypoplasia. Polymerase chain reaction for parvoviral deoxyribonucleic acid was positive in cerebellar tissue. Cell-specific immunolabeling was used to characterize the lesions, which were characterized into 2 types. In type 1 lesions, the cortex was nearly agranular, with an extremely thin molecular layer; the Purkinje cells were randomly placed and oriented, and their stunted main dendrite produced a thorn-covered atrophic dendritic tree; the basket cell axons ran randomly and had dysmorphic endings; and myelinated fibers were severely reduced in folia axes. In type 2 lesions, the cortex was hypogranular; the Purkinje cells were linearly organized, but their main dendrite extended too far in the molecular layer before giving up smooth, bent secondary dendrites; many basket cells were located along the cerebellar surface, and their axons ran at right angle to the surface; myelinated fibers were moderately reduced. Defects in climbing fiber synapse translocation and elimination were evident in both types of lesion. This immunohistologic study allowed a comparison between lesions in these spontaneous cerebellar hypoplasia cases with those documented when using silver impregnation studies after perinatal experimental cerebellar damage. Such a comparison is consistent with viral infection that occurs before birth in all 3 cases. Progress in parvovirus biology knowledge suggests that viral NS1 protein cytotoxicity might explain degenerative changes in the Purkinje cells that were present, in addition to the development defect.

Brainstem Auditory Evoked Potential Wave V Latency‐Intensity Function in Normal Dalmatian and Beagle Puppies

This study investigated whether Dalmatian puppies with normal hearing bilaterally had the same click-evoked brainstem auditory potential characteristics as age-matched dogs of another breed. Short-latency brainstem auditory potentials evoked by condensation and rarefaction clicks were recorded in 23 1.5- to 2-month-old Dalmatian puppies with normal hearing bilaterally by a qualitative brainstem auditory evoked potential test and in 16 Beagle dogs of the same age. For each stimulus intensity, from 90 dB normal hearing level down to the wave V threshold, the sum of the potentials evoked by the 2 kinds of stimuli were added, giving an equivalent to the alternate click polarity stimulation. The slope of the L segment of the wave V latency-intensity curve was steeper in Dalmatian (-40 +/- 10 micros/dB) than in Beagles (-28 +/- 5 micros/dB, P < .001) puppies. The hearing threshold was lower in the Beagle puppies (P < .05). These results suggest that interbreed differences may exist at the level of cochlear function in this age class. The wave V latency and wave V-wave I latencies differences at high stimulus intensity were different between the groups of puppies (4.3 +/- 0.2 and 2.5 +/- 0.2 milliseconds, respectively, for Beagles; and 4.1 +/- 0.2 and 2.3 +/- 0.2 milliseconds for Dalmatians, P < .05). A different maturation speed of the neural pathways is one possible explanation of this observation.

Frequency tuning curves derived from auditory steady state evoked potentials: a proof-of-concept study.

Objectives: Assess the feasibility of drawing tuning curves from the masking function of steady state potentials. Develop a noninvasive tool for research applications on cochlear frequency selectivity in sedated animals. Obtain pilot human data validating auditory steady state evoked potential-derived (ASSEP) tuning curves against psychophysical data. Design: ASSEP tuning curves were drawn in 10 Beagle puppies and six human adults using amplitude-modulated probes. Two probe frequencies (1 and 2 kHz) were used in dogs and only one (2 kHz) in humans. The modulation rates of the two probes were set to 81 and 88 Hz, respectively. Psychophysical tuning curves were obtained in 12 normal human subjects using the same maskers and either a pure-tone or an amplitude-modulated probe to verify if the latter had a specific effect on tuning curve parameters. Six of these 12 subjects participated in the electrophysiologic measurements. For each tuning curve, the intensity of the narrowband masker required just to mask the fixed probe was plotted for different masker center frequencies. Masker center frequencies extended to about half an octave above and an octave below the probe frequencies in 100-Hz steps. Tuning curve width (Q10 dB values), high- and low-frequency slopes (in dB/octave) and the masker frequency yielding the lowest masking threshold (maximal masker frequency) were computed. Canine Q10 dB values obtained were compared with those published for several species with other techniques. For humans, ASSEP and psychophysical tuning curves were directly compared in the same subjects and with published data. Results: In dogs, the ASSEP method yielded reproducible tuning curves with qualitative and quantitative parameters similar to other physiologic measures of tuning obtained in various animals. Q10 dB values were greater at 2 than at 1 kHz, reflecting the well-known correlation between sharpness of tuning and central frequency. In humans, ASSEP Q10 dB values were slightly smaller than the psychophysical ones, but were greater by a factor of 2 than those obtained with previously published electrophysiologic procedures. In both species, detuning—a shift of the tip of the curve away from the probe frequency—was frequently observed as upward shifts with a maximal value of 200 Hz. Human psychophysical tuning curves also showed a certain amount of upward detuning. The intraindividual comparison of the two types of probes performed on human subjects with the psychophysical method did not indicate a specific effect of the amplitude-modulated probe on the curve parameters. Neither did the intraindividual comparisons indicate that an amplitude-modulated probe per se promoted detuning. Detuning has been observed with several other techniques and is usually attributed to nonlinear interactions between masker and probe in simultaneous masking. Conclusions: The results demonstrate the feasibility of measuring realistic ASSEP tuning curves in sedated dogs and in sleeping human adults. The ASSEP tuning curves exhibit a series of classical features similar to those obtained with time-honored methods. These results pave the way for the development of a noninvasive electrophysiologic method for tuning curve recording and its applications in noncooperative experimental animals or clinical subjects.

Purkinje cell neuroaxonal dystrophy similar to nervous mutant mice phenotype in two sibling kittens

Three 4-month-old kittens from the same litter were presented, two of which were exhibiting cerebellar signs. Euthanasia was requested. No cerebellum atrophy was disclosed on necropsy. General cerebellar anatomy was normal, including the thickness of the cortical layers, myelination, and neurons of the deep cerebellar nuclei. In the ataxic cat vermis, Purkinje cells were lacking along broad parasagittal bands symmetrically disposed relative to the midline. Many Purkinje cells were also lacking in the hemispheres. The nodulus and the flocculus were normal. Surviving Purkinje cells had frequent main dendrite swellings visible with anti-calbindin and anti-microtubule associated protein. In affected regions, calbindin and phosphorylated neurofilaments immunesera stained numerous axonal torpedoes located in the granular layer and the folial white matter. They were also present in processes of the deep cerebellar nuclei and lateral vestibular nucleus. Loss of synaptic endings onto the neurons of these nuclei was evident. Hypertrophied Purkinje cell recurrent axons and enhanced retrograde synaptic endings were present in the granular layer. Bergmann glia was strongly labeled by anti-GFAP, but no abnormal supplementary fibers were seen. None of these alterations were present in the normal sister. However, abnormal vacuolation of the Purkinje cell main dendrites was evident in all three cats, but not in six unrelated control cats that were 3–6 months old. The inferior olive and pontine nuclei were also normal. The two ataxic cats had a primary Purkinje cell degeneration that shared many common features with the abnormal Purkinje cells of the nervous mutant mouse.

Detection of antigenic heterogeneity in feline coronavirus nucleocapsid in feline pyogranulomatous meningoencephalitis.

A new monoclonal antibody (mAb), CCV2-2, was compared with the widely used FIPV3-70 mAb, both directed against canine coronavirus (CCoV), as a diagnostic and research tool. Western blot showed that both anti-CCoV mAbs only reacted with a protein of 50 kD, a weight consistent with the feline coronavirus (FCoV) viral nucleocapsid. A competitive inhibition enzyme-linked immunosorbent assay showed that the 2 recognized epitopes are distinct. Preincubation of CCV2-2 mAb with FCoV antigen suppressed the immunostaining. Formalin-fixed, paraffin-embedded sections from brains of 15 cats with the dry form of feline infectious peritonitis (FIP) were examined by immunohistochemistry. Immunohistochemistry was performed with both anti-CCoV mAbs, either on consecutive or on the same sections. A myeloid-histiocytic marker, MAC 387, was also used to identify FIP virus-infected cells. In all regions where MAC 387-positive cells were present, positive staining with the CCV2-2 mAb was systematically detected, except at some levels in 1 cat. In contrast, none or only a few cells were positive for the FIPV3-70 mAb. Double immunostaining showed macrophages that were immunopositive for either CCV2-2 alone or alternatively for CCV2-2 and FIPV3-70 mAbs. This reveals the coexistence of 2 cohorts of phagocytes whose FIP viral contents differed by the presence or absence of the FIPV3-70-recognized epitope. These findings provide evidence for antigenic heterogeneity in coronavirus nucleocapsid protein in FIP lesions, a result that is in line with molecular observations. In addition, we provide for the first time morphologic depiction of viral variants distribution in these lesions.