Neuropathological changes in dorsal root ganglia induced by pyridoxine in dogs

doi:10.21203/rs.2.17162/v2

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Neuropathological changes in dorsal root ganglia induced by pyridoxine in dogs

https://doi.org/10.21203/rs.2.17162/v2

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published 24 Mar, 2020

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Background: Pyridoxine (PDX), vitamin B₆, is an essential vitamin. PDX deficiency induces various symptoms and abuse of PDX supplement also acts as a neurotoxicant that induces severe sensory neuropathy.

Results: To assess the possibility of reversible sensory neuropathy model using dogs, 150 mg/kg pyridoxine (PDX) was injected subcutaneously into dogs for seven days and body weight measurement, postural reaction assessments, and electrophysiological recordings were conducted. In addition, the morphology of dorsal root ganglia (DRG) and changes of glial fibrillary acidic protein (GFAP) immunoreactive satellite glial cells and ionized calcium-binding adapter molecule 1 (Iba-1) immunoreactive microglia/macrophages were observed at 0, 1 and 4 weeks after the last PDX treatment. During the administration period, body weight loss and proprioceptive loss occurred. After the treatments were completed, electrophysiological recordings showed that the H-reflex of the treated dogs had disappeared at week 0. These phenomena persisted for four weeks exceptional body weight. CV and HE staining revealed that neurons in DRG had significant loss of large-sized neurons 0 and 1 week, but these neurons were recovered 4 weeks. Iba-1 and GFAP immunohistochemistry showed that reactive microglia/macrophages and satellite glial cells were pronounced at 0 and 1 weeks after the last PDX treatment, respectively and thereafter decreased with time after PDX treatment, respectively.

Conclusions: This result suggests that PDX-induced neuropathy model is reversible and can be a good experimental model for research on neuropathy in dogs.

Computational Neuroscience

dog

dorsal root ganglia

H-reflex

pyridoxine

sensory neuropathy

Pyridoxine (PDX) is one of the compounds that can be called vitamin B₆, along with pyridoxal and pyridoxamine. PDX is an essential vitamin, and PDX deficiency induces various symptoms [1]. However, PDX has been identified as a neurotoxicant that induces severe sensory neuropathy in response to chronic abuse of oral PDX supplements [2]. Compelling evidence related to the functional and physiological parameters induced by PDX neurotoxicity has been demonstrated in rodents [3-6], dogs [7-9], and humans [10].

There are many disorders that involve invasion of the peripheral nerve, inducing sensory neuropathy, which is caused by genetic diseases, metabolic imbalance, endocrine disease, toxins, fluoroquinolone toxicity, inflammation, and physical trauma. Accordingly, many clinical trials have been conducted to treat sensory neuropathy; however, more animal models of sensory neuropathy are required [11-13]. Dogs are valuable animal models because they are more similar to humans than rodents. However, fewer neurological studies have been conducted using canine models than rodent models [7].

All sensory axons pass into the Dorsal root ganglion (DRG) and then into the spinal cord itself. Therefore, observation of DRG is useful to obtain information about the sensory tract [14].

In this study, we conducted neurological examination and observed H-reflex and chronological changes in satellite glial cells (SGC) and microglia/macrophages using glial fibrillary acidic protein (GFAP) and ionized calcium-binding adapter molecule 1 (Iba-1) in DRG, respectively. Specifically, we evaluated H-reflex and expression of GFAP and Iba-1 to determine if they changed with time in response to subcutaneous injection of PDX for seven days. To accomplish this, a dog model of sensory neuropathies was established by administering subcutaneous injection of PDX over a short term period. We observed the dogs for four weeks after the last PDX treatment to identifying chronological changes in the effects of PDX on DRG.

Body weight measurements

Weight loss was observed in the treated group (Fig. 1), with significant differences in body weight being observed at 0 (100 ± 0 %) and 1 week after the last PDX treatment (87.3 ± 3.8 %) (p < 0.0001). However, there was no significant difference in the weight between before PDX treatment (100 ± 0 %) and 4 weeks after the last PDX treatment (97.9 ± 4.8 %) (p = 0.6579).

Postural reaction assessments

All dogs in the control group showed normal response with postural reaction test. However, all dogs in the treated group developed neurologic disorders. On day 3 or 4 of PDX injection, proprioceptive abnormalities involving the hindquarters were observed based on the postural reaction test. At 0 week after the last PDX treatment, all dogs experienced hindlimb stiffness when attempting to walk or turn. At 1 week after the last PDX treatment, these phenomena were maintained. At the end of the experimental period (4 weeks after the last PDX treatment) the hindlimb stiffness had disappeared, but the proprioceptive abnormalities were maintained.

Electrophysiological recording

Evaluation of the Direct-evoked muscle potentials (DEMP; M wave) and Reflex-evoked muscle potentials (REMP; H-reflex) in clinically normal dogs revealed the following. The mean M wave amplitudes (5.4 ± 2.3 mV) were much higher than the mean H-reflex amplitudes (0.5 ± 0.3 mV). The mean M wave latencies (2.8 ± 0.3 ms) showed that the M wave was an early response; however, the mean H-reflex latencies (16.2 ± 2.5 ms) showed that the H-reflex was a late response [6]. In the treated group, there were no significant differences in M wave amplitude at 0 week (5.6 ± 0.9 mV), 1 week (5.8 ± 1.3 mV) or 4 weeks (5.9 ± 0.9 mV) after the last PDX treatment when compared with before PDX treatment (p>0.05) (Fig.2A). However, there were significant differences in the H-reflex amplitude at 0 week, 1 week and 4 weeks after the last PDX treatment when compared with before PDX treatment (p<0.05) (Fig.2B). At 0 week, the H-reflex of the treated dogs had disappeared, and this condition was sustained until 4 weeks after the last PDX treatment. Interestingly, just one dog among three dogs, two times of H-reflex had appeared among 8 times measurement.

Immunohistochemistry

In control dogs, abundant neurons were observed in all levels of DRGs based on cresyl violet (CV) (Fig. 3A, 3E and 3I) and Hematoxylin and eosin (HE) (sFig. 1A, 1E and 1I) staining. At 0 week after the last PDX treatment, the number of CV and HE stained large-sized neurons was significantly decreased in the cervical, thoracic and lumbar level of DRG (Fig. 3M), while CV and HE stained small- and medium-sized neurons were intact. In addition, there were no significant differences in neuronal damage between regions of DRG (Fig. 3B, 3F, 3J and 3M). However, many HE stained nuclei were observed in all levels of DRGs (sFig. 1B, 1F and 1J).

At 1 week after the last PDX treatment, the number of CV stained neurons in large-sized neurons was significantly increased in all levels of DRG compared to that in the group at 0 week after the last PDX treatment and about 77.3% of those in the control group (Fig. 3C, 3G, 3K and 3M). In addition, HE staining in this group revealed that some hematoxylin stained nuclei were aggregated in the cervical levels of DRG (sFig. 1C), not in the thoracic and lumbar levels (sFig. 1G and 1K). At 4 weeks after the last PDX treatment, the number of CV stained large-sized neurons was increased in all levels of DRG compared to that in the groups at 0 or 1 weeks after the last PDX treatment, and about 92.3% neurons were detected in this group when compared to the control group (Fig. 3D, 3H, 3L and 3M). In addition, HE staining in this group showed less aggregated nuclei stained with hematoxylin in all levels of DRGs (sFig. 1D, 1H and 1L).

GFAP immunoreactive SGCs were detected in all levels of DRG in the control group, and this surrounded the neurons in the DRG (Fig. 4A, 4E and 4I). At 0 week after the last PDX treatment, GFAP immunoreactivity was slightly, but not significantly, increased in the DRG (Fig. 4B, 4F, 4J and 4M). At 1 week after PDX treatment, GFAP immunoreactivity was significantly increased in cervical, thoracic and lumbar levels of DRG when compared to those in control group, respectively (Fig. 4C, 4G, 4K and 4M). At 4 weeks after PDX treatment, GFAP immunoreactivity had decreased significantly in the all levels of DRG and was similar to those in the control group (Fig. 4D, 4H, 4L and 4M).

Ionized calcium-binding adapter molecule 1 (Iba-1) immunoreactive microglia/macrophages in control group were detected in all levels of DRG (Fig. 5A, 5E and 5I). At 0 week after the last PDX treatment, Iba-1 immunoreactivity was significantly higher in the DRG and the Iba-1 immunoreactive microglia/macrophage had hypertrophied cytoplasm with round shape (Fig. 5B, 5F, 5J and 5M). At 1 and 4 weeks after the last PDX treatment, Iba-1 immunoreactivity was gradually decreased in the DRG with time after cessation of PDX treatment, but Iba-1 immunoreactivity in these groups was higher than in the all levels of control group (Fig. 5C, 5D, 5G, 5H, 5K, 5L and 5M).

Neuropathy is disruptions in synaptic transmission between central and peripheral nervous system in the body. PDX is well known for an agent in peripheral neuropathy because it was safe for other organs [15-17]. Several studies demonstrate PDX causes neuronal death in the cells bodies and long myelinated fibers of DRGs in human, rat, and dogs [3-10]. Rat model administered 800 mg/kg/ml pyridoxine with intraperitoneal route and shows significant decreases in large-sized cells [18] and H-reflex was measured to demonstrate that PDX induced sensory neuropathy. In contrast, dog model only treated 150 mg/kg pyridoxine subcutaneously and electrophysiological recordings taken during peripheral nerve stimulation in the experimental group were consistent with selective toxicity to sensory, but not motor nerve function [7]. In the present study, we selected PDX for the induction of peripheral neuropathy in dogs because dog model is induced by low dosage of PDX compared to that in the rats. In this study, we investigated the effect of PDX on cell morphology, SGCs, and microglia/macrophages at 0, 1 and 4 weeks after the last PDX treatment on dogs to assess the possibility of reversible animal model for PDX-induced neuropathy in dogs.

Based on previous studies [7, 8], we were concerned that the changes and the relationship with time of neurological examination, electrophysiological recording, and histopathological analysis of dog model of sensory neuropathies, by PDX over a short term period. In previous experiments, body weight decreased during the PDX administration period and was related to food consumption. This phenomenon was the same for the PDX administration period, but the body weight increased with time after PDX administration in this study. We also confirmed that the H-reflex disappeared for four weeks after the last PDX treatment, even though histological analysis showed that the DRG were recovered. Based on these data, we supposed that the structural recovery of DRG occurs before its functional recovery.

In this study, we also observed the chronological changes in SGCs and microglia because these cells contribute to several neuropathological conditions [19, 20]. GFAP immunoreactivity in SGCs were significantly upregulated at 1 week after the last PDX treatment and decreased 4 weeks after the last PDX treatment. These results were similar to the previous findings that GFAP expression was upregulated in SGCs of the injured DRG from day 3 onwards immediately after sciatic nerve injury because we also observed proprioceptive abnormalities at 3-4 days after PDX treatment.

Iba-1 immunoreactive microglia were significantly increased at 0 week after the last PDX treatment and Iba-1 immunoreactive microglia continued to decrease throughout the experiment. This result was supported by our histological findings that neuronal death was prominent at 0 week after the last PDX treatment, and that the number of large-sized neurons in the DRG increased thereafter. Activated microglia/macrophages may be associated with initiation and maintenance of the mechanical hypersensitivity [21]. In diabetic neuropathy model, Iba-1 immunoreactive macrophages are significantly increased in the lumbar DRG 1 week after streptozotocin treatment [22]. In the sciatic nerve injury model, macrophages are gradually increased by 7 days after injury [23].

We established a reversible dog model of sensory neuropathies by administering subcutaneous injections of PDX over a short term period. However, further studies involving the mechanism of sensory neuropathy of PDX in dogs are necessary.

Animal model

Eleven beagle dogs approximately 2 years of age were used to evaluate PDX-induced neuropathy. The body weight of the dogs ranged from 7 to 11 kg. Two dogs were in the control group and nine were in the experimental group. Three of the dogs in the experimental group were included in the 0 week group, while three were included in the 1 week group, and three were in the 4 weeks group (based on the time after the last PDX treatment).

All dogs were clinically judged to be in good health and neurologically normal. All experimental dogs had their own admission number (SNU-200908-18) from the Institute of Laboratory Animal Resources, Seoul National University (Korea). During the experiment, all dogs were cared for according to the Animal Care and Use Guidelines of the Institute of Laboratory Animal Resources, Seoul National University.

PDX intoxication

PDX (Sigma, St. Louis, MO) was diluted in a 0.9% sterile aqueous solution of sodium chloride and administered subcutaneously (SC) once a day during the morning for seven days. PDX solution was prepared immediately prior to each injection. Animals in the control group received vehicle (iso-osmotic sterile aqueous solution of sodium chloride), while animals in the experimental group were administered 150 mg/kg PDX in a volume of 100 mg/ml SC [7].

Body weight measurements and postural reaction assessments

The body weights of the test dogs were measured every morning during the PDX injection period, as well as 1 weeks and 4 weeks after the last PDX treatment.

Postural reaction assessments (Table 1) were conducted on all the dogs every morning during the PDX injection period, as well as on one and four weeks after the last PDX treatment.

Electrophysiological recording

All dogs were pre-anesthetized with atropine (0.1 mg/kg of body weight, IM). Anesthesia was induced with diazepam and maintained with isoflurane (Baxter Healthcare, Deerfield, IL) through a semiclosed system. The subcutaneous temperature was maintained at 37-38ºC. Neuropack2 (Nihon Koden, Tokyo, Japan) was used for all recordings. All measurements were conducted in the left hindlimb of each dog.

DEMP, M wave, were recorded for the tibial nerve using 1 Hz, 0.5 ms, supramaximal stimulus. Stimulating electrodes were positioned in the distal tibial nerve and recording electrodes were positioned in the plantar interosseous muscle, with a ground electrode positioned between the stimulating electrode and recording electrode. The recording electrode was a bipolar needle electrode. REMP, H-reflex, were recorded using 1 Hz, 0.5 ms, submaximal stimulus. The stimulating electrode was positioned in the tibial nerve adjacent to the hook and a recording electrode and ground electrode were positioned in the same site of the tibial nerve where the M wave was measured. All measurements were conducted at least eight times [7]. Electrophysiological recordings were conducted before PDX treatment and at 0, 1, and 4 weeks after the last PDX treatment.

Immunohistochemistry

For euthanasia, cephalic veins of the dogs were selected for intravenous (IV) catheterization. The dogs were anesthetized with a high dose of propofol (5mg/kg of body weight, IV). and tiletamine/zolazepam (10mg/kg of body weight, IV). After confirmation of a deep anesthesia, they were perfused transcardially with 0.1 M phosphate-buffered saline (PBS, pH 7.4) and followed by 4% paraformaldehyde in 0.1 M phosphate-buffer (PB, pH 7.4). The DRGs of cervical, thoracic, lumbar spinal cords were removed and postfixed in the same fixative for 12 h, after which they were dehydrated with graded concentrations of alcohol for embedding in paraffin. Next, the paraffin-embedded tissues were sectioned on a microtome (Leica, Wetzlar, Germany) into 3-μm coronal sections and then mounted onto silane-coated slides. To elucidate the PDX-induced DRG damage, the samples were deparaffinized in xylene, rehydrated in a descending ethanol series and stained with hematoxylin and eosin. In addition, CV staining was conducted as previously described [2]. Briefly, CV acetate (Sigma) was dissolved at 1.0% (w/v) in distilled water, and glacial acetic acid was then added to this solution. Before and after staining for 2 min at room temperature, the sections were washed twice in distilled water, dehydrated with graded concentrations of alcohol at room temperature, and finally mounted with Canada balsam (Kanto, Tokyo, Japan). Images of all CV stained neurons were taken from the DRG through a BX51 light microscope (Olympus, Tokyo, Japan) equipped with a digital camera (DP71, Olympus) connected to a PC monitor. DRG neurons were separated into three categories according to their sizes: small- (area 1000 μm²), medium- (area 1000-2000 μm²), and large-sized (>2000 μm²) neurons. The number of these neurons in all the groups was counted in the DRG using an image analyzing system equipped with a computer-based CCD camera (software: Optimas 6.5, CyberMetrics, Scottsdale, AZ). Cell counts were obtained by averaging the counts from the sections taken from each animal. Immunohistochemistry for GFAP and Iba-1 was conducted to elucidate chronological changes in reactive gliosis after PDX treatment. The sections were sequentially treated with 0.3% hydrogen peroxide in PBS for 30 min and 10% normal goat serum in 0.05 M PBS for 30 min, after which they were incubated with diluted rabbit anti-GFAP (1:1000, Chemicon, Temecula, CA) or rabbit anti-Iba-1 antibody (1:500, Wako, Osaka, Japan) overnight at room temperature and subsequently exposed to FITC-conjugated anti-rabbit IgG (1:200; Jackson ImmunoResearch, West Grove, PA) and FITC-conjugated anti-rat IgG (1:600; Jackson ImmunoResearch). The immunoreactions were observed under a BX51 microscope attached to a fluorescent lamp. Analysis of a region of interest (ROI) in each levels of DRG was performed using an image analysis system. Images were calibrated into an array of 512 × 512 pixels corresponding to a tissue area of 140 × 140 μm (40× primary magnification). Each pixel resolution was 256 gray levels. The intensity of GFAP and Iba-1 immunoreactivity was evaluated by means of a relative optical density (ROD), which was obtained after the transformation of the mean gray level using the formula: ROD = log (256/mean gray level). ROD of background was determined in unlabeled portions and the value subtracted for correction, yielding high ROD values in the presence of preserved structures and low values after structural loss using NIH Image 1.59 software. A ratio of the ROD was calibrated as % .

Statistical analysis

All data are expressed as the mean ± SE or the mean ± SEM. The data were evaluated by a student’s t-test. Statistical significance was considered at P < 0.05. A paired t-test was conducted for analysis of the body weights and the M wave and H-reflex amplitudes before and after the pharmacologic treatment. The level of significance was set at p < 0.05.

Ethics approval and consent to participate

All procedures for animal care were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the by the Institutional Animal Care and Use Committee of Seoul National University (SNU-200908-18).

Consent to publish

Not applicable.

Availability of data and materials

The datasets used and/or analyzed during the current study including the tissue samples of dorsal root ganglion generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests

The authors declare no conflict of interest.

Funding

This work was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (NRF-2013R1A1A2059364, NRF-2014R1A1A1004339) and by the Promising-Pioneering Researcher Program through Seoul National University (SNU) in 2015. The funding body played no role in the design and interpretation of the experiments. The founding sponsors had no role in the study design, performance, data collection and analysis, decision to publish, or preparation/writing of the manuscript.

Authors’ Contributions

SY and WK wrote the paper. MSK, THK and YK performed the experiments. JOA and JHC analyzed the data. IKH and JYC wrote the paper, conceived and designed the experiments. All authors read and approved the final manuscript.

Acknowledgements

Not applicable.

CV: cresyl violet; DEMP: Direct-evoked muscle potentials; DRG: dorsal root ganglia; GFAP: glial fibrillary acidic protein; HE: Hematoxylin and eosin; Iba-1: ionized calcium-binding adapter molecule 1; PDX: pyridoxine; REMP: Reflex-evoked muscle potentials; ROD: relative optical density; ROI: region of interest; SGC: satellite glial cells

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Table 1. Postural reaction test

wheel-barrowing test

Hopping test

Hemi-walking test

proprioceptive positioning test

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Neuropathological changes in dorsal root ganglia induced by pyridoxine in dogs

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