Growth-associated Protein GAP-43 Research Articles

Lentivirus-mediated Knockdown of Ski Improves Neurological Function After Spinal Cord Injury in Rats.

The glial scar that forms at the site of injury after spinal cord injury (SCI) is an important physical and biochemical barrier that prevents axonal regeneration and thus delays functional recovery. Ski is a multifunctional transcriptional co-regulator that is involved in a wide range of physiological and pathological processes in humans. Previous studies by our group found that Ski is significantly upregulated in the spinal cord after in vivo injury and in astrocytes after in vitro activation, suggesting that Ski may be a novel molecule regulating astrocyte activation after spinal cord injury. Further studies revealed that knockdown or overexpression intervention of Ski expression could significantly affect the proliferation and migration of activated astrocytes. To further verify the effect of knockdown of Ski expression in vivo on glial scar formation and neurological function after spinal cord injury, we prepared a rat spinal cord injury model using Allen's percussion method and used lentivirus as a vector to mediate the downregulation of Ski in the injured spinal cord. The results showed that knockdown of Ski expression after spinal cord injury significantly suppressed the expression of glial fibrillary acidic protein (Gfap) and vimentin, hallmark molecules of glial scarring, and increased the expression of neurofilament protein-200 (Nf-200) and growth-associated protein (Gap43), key molecules of axon regeneration, as well as Synaptophysin, a key molecule of synapse formation expression. In addition, knockdown of Ski after spinal cord injury also promoted the recovery of motor function. Taken together, these results suggest that Ski is able to inhibit the expression of key molecules of glial scar formation, and at the same time promotes the expression of molecules that are markers of axonal regeneration and synapse formation after spinal cord injury, making it a potential target for targeted therapy after spinal cord injury.

Design and construction of a GAP-43 reporter system for potential identification of effective therapeutics for peripheral nerve regeneration

Background: Peripheral nerve injury (PNI) is a condition that can result in muscle paralysis and sensory disturbances. Electrical stimulation and/or the application of exogenous neurotrophic factors and cytokines are effective at enhancing nerve regeneration and is mediated via the expression of regeneration associated genes (RAGs) such as the growth associated protein GAP-43. Therapeutic upregulation of GAP-43 has potential use as a treatment for improving recovery from PNI. Few studies have investigated the potential of increasing GAP-43 for PNI therapeutic purposes and current methods for measuring GAP-43 expression are limited. Aims and Objectives: The broader aim of this work was to construct a motor neuron-like cell model with a GAP-43 reporter system. Such a model would have potential use in screening for novel therapeutics that upregulate GAP-43 and in the optimisation of electrical stimulation treatment in combination with these therapies. The key aim of the work was to design and construct a Cas9 expressing plasmid bearing a gRNA that targets GAP-43 cleavage and a donor plasmid bearing a reporter GFP or Neo cassette flanked with 5’ and 3’ GAP-43 homology arms (HAs) to facilitate the insertion of the reporter immediately 3’ of the GAP-43 promoter via CRISPR/Cas9 homology directed repair (HDR). Such an insertion would enable quantitative measurement of endogenous expression of the GAP-43 gene. Methods and Results: To guide the Cas9 nuclease to the target location, GAP-43 gRNA oligomers were designed and cloned downstream of the U6 promoter in the Cas9 expression plasmid px330, which also expresses the Cas9 gene and the cloned gRNA when transfected into cells. For CRISPR/Cas9 HDR, the 5’ and 3’ GAP-43 HAs were amplified from mouse genomic DNA and cloned into the donor plasmid using Gibson Assembly so that they flanked the reporter cassette. Results: This work successfully constructed the Cas9 gRNA expressing plasmid to target cleavage of the GAP-43 gene in mouse cell lines and has provided the complete design and construction foundation for generation of the reporter system for the endogenous GAP-43 gene in mice.

Development of Neurogenic Detrusor Overactivity after Thoracic Spinal Cord Injury Is Accompanied by Time-Dependent Changes in Lumbosacral Expression of Axonal Growth Regulators.

Thoracic spinal cord injury (SCI) results in urinary dysfunction, which majorly affects the quality of life of SCI patients. Abnormal sprouting of lumbosacral bladder afferents plays a crucial role in this condition. Underlying mechanisms may include changes in expression of regulators of axonal growth, including chondroitin sulphate proteoglycans (CSPGs), myelin-associated inhibitors (MAIs) and repulsive guidance molecules, known to be upregulated at the injury site post SCI. Here, we confirmed lumbosacral upregulation of the growth-associated protein GAP43 in SCI animals with bladder dysfunction, indicating the occurrence of axonal sprouting. Neurocan and Phosphacan (CSPGs), as well as Nogo-A (MAI), at the same spinal segments were upregulated 7 days post injury (dpi) but returned to baseline values 28 dpi. In turn, qPCR analysis of the mRNA levels for receptors of those repulsive molecules in dorsal root ganglia (DRG) neurons showed a time-dependent decrease in receptor expression. In vitro assays with DRG neurons from SCI rats demonstrated that exposure to high levels of NGF downregulated the expression of some, but not all, receptors for those regulators of axonal growth. The present results, therefore, show significant molecular changes at the lumbosacral cord and DRGs after thoracic lesion, likely critically involved in neuroplastic events leading to urinary impairment.

Effects of electroacupuncture combined with olfactory ensheathing cell transplantation on growth associated protein GAP-43 levels in the injured spinal cord region

Purpose: The study of electro-acupuncture combined with olfactory ensheathing cells ( OECs ) transplantation for treatment of rats with spinal cord injury ( SCI ) of spinal motor function recovery, pathological change and the relationship between them. In order to determine the effect of the combination of the two, and whether a single processing factors can make the axonal regeneration and the regeneration of axons have oriented sex effect, so that the spinal cord function recovered to a certain extent, electroacupuncture and olfactory ensheathing cells in repair of spinal cord injury process produces a synergistic effect and its mechanism of action.

Restored presynaptic synaptophysin and cholinergic inputs contribute to the protective effects of physical running on spatial memory in aged mice

The effects of prolonged physical training on memory performance and underlying presynaptic mechanisms were investigated in old C57BL/6 mice. Training via voluntary running wheels was initiated at 16 months of age and continued for 5 months (1 h per day, 5 days per week), followed by testing of learning and memory functions and counting of presynaptic puncta and cholinergic inputs in the hippocampus. Trained old mice were compared to their age-matched sedentary controls and adult controls. This training strategy improved hippocampal-dependent spatial memory function tested via a novel location task, and enhanced memory was accompanied by restored presynaptic puncta and cholinergic fibers in area CA1 and DG of the hippocampus in old mice. Particularly, the training selectively affected presynaptic vesicle protein synaptophysin but not growth associated protein GAP-43, and the increased number of synaptophysin puncta positively correlates with improved memory performance. To better understand the neurochemical mechanisms by which prolonged physical training protects against aging-related memory deficits, the cholinergic inputs to the hippocampus were compared among the three groups of mice and correlated with memory performance. While the running prevented age-related loss of cholinergic inputs, it has limited impact on the projection source cells in the medial septum-diagonal band (MS-DB). Importantly, cholinergic fibers in area CA1 and DG positively correlated with spatial memory function. These data suggest that the preservation of presynaptic inputs, particularly those involved in the integrity of memory performance, contributes critically to the beneficial effects of physical running initiated at an older age.

Mitotic activity, modulation of DNA processing, and purinergic signalling in the adult rat auditory brainstem following sensory deafferentation.

A complex scenario of cellular network reorganization is caused by unilateral sensory deafferentation (USD) in the adult rat central auditory system. We asked whether this plasticity response involves mitosis. Immunohistochemistry was applied to brainstem sections for the detection and localization of mitotic markers Ki67 and PCNA, the growth-associated protein Gap43 and purine receptor P2X4. Fluorescent double staining was done for Ki67:PCNA and for both of them with HuC/HuD (neurons), S100 (astrocytes), Iba1 (microglia) and P2X4. Inquiring 1-7days after USD, we found Ki67 expression to be changed in cellular profiles of cochlear nucleus (CN) with a significant increase in number by 1-3days, followed by reset to control level within 1week. USD-induced mitosis exclusively occurred in microglia and was absent elsewhere in the auditory brainstem. PCNA staining of small cellular profiles increased similarly but remained elevated. PCNA staining intensity also changed in CN, superior olive and inferior colliculus in neuronal nuclei, suggesting shifts in DNA processing. No apoptotic cell death was detected in any region of the adult auditory brainstem after USD. A comparison of anterograde and retrograde effects of nerve damage revealed proliferating microglia expressing P2X4 receptors in CN upon USD, but not in the facial nucleus after facial nerve transection. In conclusion, the deafferentation model studied here permits insight into the capacity of the adult mammalian brain to invoke mitosis among glia cells, adjustment of gene processing in neurons and purinergic signalling between them, jointly accounting for a multilayered neuro- and glioplastic response.

Neuroregenerative Effects of Electromagnetic Field and Magnetic Nanoparticles on Spinal Cord Injury in Rats.

The present study aimed to evaluate the effect of iron oxide nanoparticles (IONPs) along with electromagnetic fields (MF) exposure on spontaneous and induced axonal sprouting after spinal cord injury (SCI). Adult male Wistar rats were subjected to spinal cord transection at the T13 segment. The IONP (25 μg/mL) embedded in 3% agarose gel was implanted at the injury site and subsequently exposed to MF (50 Hz, 17.96 μT, 2 hours/day for 5 weeks). Histological analysis of spinal cord tissue showed a significant increase in the expression of the growth-associated protein GAP-43 and it was found to be co-localized with neuronal nuclei marker and neurofilaments. The results show sprouting from mature neurons and axons, significantly less demyelination and more myelinated fibers were evident at the lesion site. However, no motor or somatosensory evoked potential response was observed, suggesting lack of long-distance functional connectivity. These findings highlight the therapeutic potential of IONPs along with MF exposure in promoting neuroregeneration after SCI.

Modulation of hippocampal neuronal activity by So-ochim-tang-gamibang in mice subjected to chronic restraint stress

BackgroundSo-ochim-tang-gamibang (SOCG) is a decoction formula which has been used to improve mental activity in traditional Korean medicine. The present study was performed to evaluate whether the treatment of SOCG was involved in activating hippocampal neurons in mice which were subjected to chronic restraint stress (CRS).MethodsMice were subjected to CRS for 2 weeks to induce depressive-like behaviors. SOCG was orally administered for the same period. mRNA expression in the hippocampus was analyzed by RT-PCR. Levels of serotonin receptor 5-HT1AR in the hippocampus were determined by western blotting and by immunofluorescence staining in coronal brain sections. Cultured neurons were prepared from the dorsal root ganglia (DRG) in mice to examine the effects of CRS and SOCG treatment on neurite outgrowth. Depressive-like behaviors of experimental animals were measured by open field test (OFT) and forced swimming test (FST).ResultsmRNA levels of serotonin 1A and 1B receptors (5-HT1AR and 5-HT1BR) were decreased in the hippocampus of CRS animals and increased by SOCG treatment. Signals of 5-HT1AR protein in CA3 pyramidal cells were decreased by CRS but elevated back to levels in control animals after SOCG treatment. Phospho-Erk1/2 protein in CA3 cells showed similar pattern of changes as in 5-HT1AR, suggesting coordinated regulation after SOCG treatment in CRS animals. Axonal growth-associated protein GAP-43 levels were also decreased by CRS and then increased by SOCG treatment. In vivo administration of SOCG improved neurite outgrowth of primary DRG neurons from CRS animals and also increased 5-HT1AR protein signals. Behavioral tests of open field and forced swimming showed that immobility time periods were significantly decreased by SOCG treatment.ConclusionsOur data suggest that SOCG treatment may increase synaptic responsiveness to serotonergic neuronal inputs by upregulating 5-HT1AR in the hippocampal neurons.

BDNF gene delivery mediated by neuron-targeted nanoparticles is neuroprotective in peripheral nerve injury.

Neuron-targeted gene delivery is a promising strategy to treat peripheral neuropathies. Here we propose the use of polymeric nanoparticles based on thiolated trimethyl chitosan (TMCSH) to mediate targeted gene delivery to peripheral neurons upon a peripheral and minimally invasive intramuscular administration. Nanoparticles were grafted with the non-toxic carboxylic fragment of the tetanus neurotoxin (HC) to allow neuron targeting and were explored to deliver a plasmid DNA encoding for the brain-derived neurotrophic factor (BDNF) in a peripheral nerve injury model. The TMCSH-HC/BDNF nanoparticle treatment promoted the release and significant expression of BDNF in neural tissues, which resulted in an enhanced functional recovery after injury as compared to control treatments (vehicle and non-targeted nanoparticles), associated with an improvement in key pro-regenerative events, namely, the increased expression of neurofilament and growth-associated protein GAP-43 in the injured nerves. Moreover, the targeted nanoparticle treatment was correlated with a significantly higher density of myelinated axons in the distal stump of injured nerves, as well as with preservation of unmyelinated axon density as compared with controls and a protective role in injury-denervated muscles, preventing them from denervation. These results highlight the potential of TMCSH-HC nanoparticles as non-viral gene carriers to deliver therapeutic genes into the peripheral neurons and thus, pave the way for their use as an effective therapeutic intervention for peripheral neuropathies.

Enhanced axonal regeneration of the injured sciatic nerve by administration of Buyang Huanwu decoction

Ethnopharmacological relevanceBuyang Huanwu decoction (BYHWD) has been used in the traditional Chinese medicine for the treatment of cardiovascular and neurological symptoms, and recent experimental studies have begun to provide evidence showing its protective effects on neural cells. Yet, its function for the regenerative responses of axons in the peripheral nerve after injury is not known. Aim of the studyThe primary objective of the present study was to explore that BYHWD is involved in growth-promoting activity of the peripheral nerve axons after injury. We further examined whether the effect of BYHWD exerted directly on regrowing axons or Schwann cells. Materials and methodsSciatic nerves in rats were given crush injury, and BYHWD was injected by oral administration. Sciatic nerves or DRG tissues were prepared for immunofluorescence staining and western blot analysis. Levels of axonal regeneration were quantified by retrograde tracing technique. Cultured DRG sensory neurons and Schwann cells were prepared from rats and used to examine the effects of BYHWD on the neurite outgrowth. Behavioral analysis on functional recovery after nerve injury was assessed in mice by pin prick test, adhesive removal test, and toe-spreading reflex. ResultsImmunofluorescence and retrograde tracing analyses showed that the distal extension of the sciatic nerve axons was significantly improved by BYHWD treatment. Levels of axonal growth-associated protein GAP-43 were upregulated by BYHWD treatment in the sciatic nerve after injury and in the neurites of cultured DRG neurons. In vivo administration of BYHWD in rats upregulated the induction level of cell division cycle 2 (Cdc2) and its phosphorylation of vimentin in Schwann cells from injured sciatic nerve. Coculture of DRG neurons with Schwann cells prepared from preinjured sciatic nerves in animals administered with BYHWD led to the enhancement in neurite outgrowth. Behavioral tests in mice given sciatic nerve injury showed a significant improvement in sensorimotor activity by BYHWD administration. ConclusionsOur results suggest that BYHWD administration into animals given sciatic nerve injury facilitates axonal regeneration by acting on both the axons undergoing regeneration and neighboring Schwann cells and improves functional recovery.

Ligustilide prevents cognitive impairment and attenuates neurotoxicity in d-galactose induced aging mice brain

Ligustilide (LIG) is a principal active ingredient of traditional Chinese medicine, Radix Angelica sinensis, which has versatile pharmacological activities including neuroprotection. Previous studies have demonstrated that LIG has beneficial effects on cognition deficits associated with cerebral damage or neurodegenerative disorders. In present study, we investigated the neuroprotective effect of LIG on cognitive impairment and neurotoxicity in the brain of aging mouse induced by d-galactose (d-gal). The aging model mice were induced by subcutaneous (S.C.) injection of d-gal once daily for 8 weeks and LIG (80mg/kg) was simultaneously administered orally. The Morris water maze (MWM) test was used to assess the spatial learning and memory abilities. The activity of Na+–K+-ATPase and the content of lipid peroxidation product malondialdehyde (MDA) in brain were examined. The levels of glial fibrillary acidic protein (GFAP), growth-associated protein GAP-43, and cleaved caspase-3 in brain were also determined by immunohistochemistry. The MWM test showed that LIG administration markedly improved behavioral performance of d-gal treated mice. This action could be partly explained by the results that LIG reduced the level of MDA as well as increased the activity of Na+–K+-ATPase in the brain of d-gal induced aging mice. Moreover, LIG significantly raised the expression of GAP-43 and reduced cleaved caspase-3 and GFAP levels in the brain of d-gal treated mice. These results demonstrated that LIG improves d-gal-induced cognitive dysfunction and brain toxicity, which suggests that LIG may be developed as a new medicine for the treatment of aged-related conditions.

GAP-43 expression correlates with spinal motoneuron regeneration following root avulsion

BackgroundThe growth-associated protein GAP-43 plays a crucial role in axonal regeneration in injured neurons.MethodsWe have used immunohistochemistry to investigate the expression of GAP-43 in spinal motoneurons during nerve reconstruction following root avulsion in the neonatal and adult rats.ResultsFollowing the injury, GAP-43-immunoreactivity (IR) could be found in adult avulsed motoneurons as early as 1 day, increased from 3 to 7 days and reached a maximal level at 2 weeks post-injury. The up-regulation of GAP-43 in adult avulsed motoneurons was accompanied with the axonal regeneration indicated by numerous regenerating motor axons entering the reimplanted ventral root and nerve. In contrast, GAP-43-IR could not be found in the neonatal avulsed motoneurons at any examined post-injury time points. This failure of up-regulation of GAP-43 was coincident with no axonal regeneration in the reimplanted nerve in the neonatal rats.ConclusionClose association of GAP-43 expression and capacity of regeneration in reimplanted spinal nerve of avulsed motoneurons suggests that GAP-43 is a potential therapeutic target for treatment of root avulsion of brachial plexus.

Interplay between phosphorylation and palmitoylation mediates plasma membrane targeting and sorting of GAP43.

Phosphorylation and lipidation provide posttranslational mechanisms that contribute to the distribution of cytosolic proteins in growing nerve cells. The growth-associated protein GAP43 is susceptible to both phosphorylation and S-palmitoylation and is enriched in the tips of extending neurites. However, how phosphorylation and lipidation interplay to mediate sorting of GAP43 is unclear. Using a combination of biochemical, genetic, and imaging approaches, we show that palmitoylation is required for membrane association and that phosphorylation at Ser-41 directs palmitoylated GAP43 to the plasma membrane. Plasma membrane association decreased the diffusion constant fourfold in neuritic shafts. Sorting to the neuritic tip required palmitoylation and active transport and was increased by phosphorylation-mediated plasma membrane interaction. Vesicle tracking revealed transient association of a fraction of GAP43 with exocytic vesicles and motion at a fast axonal transport rate. Simulations confirmed that a combination of diffusion, dynamic plasma membrane interaction and active transport of a small fraction of GAP43 suffices for efficient sorting to growth cones. Our data demonstrate a complex interplay between phosphorylation and lipidation in mediating the localization of GAP43 in neuronal cells. Palmitoylation tags GAP43 for global sorting by piggybacking on exocytic vesicles, whereas phosphorylation locally regulates protein mobility and plasma membrane targeting of palmitoylated GAP43.

Increased expression of GAP43 in interneurons in a rat model of experimental polymicrogyria.

To investigate seizure susceptibility in polymicrogyria, the seizure threshold and growth-associated protein GAP43 expression were analyzed in a rat experimental model of polymicrogyria induced by intracerebral injection of ibotenate. A total of 72 neonates from 9 pregnant rats were used. Intraperitoneal pentylenetetrazole injection did not induce any seizure activity in the control rats, although it elicited seizures of variable severity in the polymicrogyria rats. Fluoro-Jade B-positive degenerating interneurons were found in the polymicrogyria brains; however, no such neurons were detected in the control brains. In the polymicrogyria rats, the GAP43 expression was significantly and widely distributed in the brain, and the percentage of parvalbumin-positive interneurons in the GAP43-positive cells was significantly higher than that observed in the nonphosphorylated neurofilament-positive pyramidal cells. We conclude that the relatively selective vulnerability of inhibitory interneurons constitutes the basis for the decreased seizure threshold observed in this model of polymicrogyria.

Inosine improves functional recovery after experimental traumatic brain injury

Despite years of research, no effective therapy is yet available for the treatment of traumatic brain injury (TBI). The most prevalent and debilitating features in survivors of TBI are cognitive deficits and motor dysfunction. A potential therapeutic method for improving the function of patients following TBI would be to restore, at least in part, plasticity to the CNS in a controlled way that would allow for the formation of compensatory circuits. Inosine, a naturally occurring purine nucleoside, has been shown to promote axon collateral growth in the corticospinal tract (CST) following stroke and focal TBI. In the present study, we investigated the effects of inosine on motor and cognitive deficits, CST sprouting, and expression of synaptic proteins in an experimental model of closed head injury (CHI). Treatment with inosine (100mg/kg i.p. at 1, 24 and 48h following CHI) improved outcome after TBI, significantly decreasing the neurological severity score (NSS, p<0.04 vs. saline), an aggregate measure of performance on several tasks. It improved non-spatial cognitive performance (object recognition, p<0.016 vs. saline) but had little effect on sensorimotor coordination (rotarod) and spatial cognitive functions (Y-maze). Inosine did not affect CST sprouting in the lumbar spinal cord but did restore levels of the growth-associated protein GAP-43 in the hippocampus, though not in the cerebral cortex. Our results suggest that inosine may improve functional outcome after TBI.

Erratum to: Palmitoylcarnitine Affects Localization of Growth Associated Protein GAP-43 in Plasma Membrane Subdomains and its Interaction with Gαo in Neuroblastoma NB-2a Cells

Erratum to: Palmitoylcarnitine Affects Localization of Growth Associated Protein GAP-43 in Plasma Membrane Subdomains and its Interaction with Gαo in Neuroblastoma NB-2a Cells

Structural plasticity of climbing fibers and the growth-associated protein GAP-43

Structural plasticity occurs physiologically or after brain damage to adapt or re-establish proper synaptic connections. This capacity depends on several intrinsic and extrinsic determinants that differ between neuron types. We reviewed the significant endogenous regenerative potential of the neurons of the inferior olive (IO) in the adult rodent brain and the structural remodeling of the terminal arbor of their axons, the climbing fiber (CF), under various experimental conditions, focusing on the growth-associated protein GAP-43. CFs undergo remarkable collateral sprouting in the presence of denervated Purkinje cells (PCs) that are available for new innervation. In addition, severed olivo-cerebellar axons regenerate across the white matter through a graft of embryonic Schwann cells. In contrast, CFs undergo a regressive modification when their target is deleted. In vivo knockdown of GAP-43 in olivary neurons, leads to the atrophy of their CFs and a reduction in the ability to sprout toward surrounding denervated PCs. These findings demonstrate that GAP-43 is essential for promoting denervation-induced sprouting and maintaining normal CF architecture.

Palmitoylcarnitine Affects Localization of Growth Associated Protein GAP-43 in Plasma Membrane Subdomains and its Interaction with Gαo in Neuroblastoma NB-2a Cells

Palmitoylcarnitine was observed previously to promote differentiation of neuroblastoma NB-2a cells, and to affect protein kinase C (PKC). Palmitoylcarnitine was also observed to increase palmitoylation of several proteins, including a PKC substrate, whose expression augments during differentiation of neural cells-a growth associated protein GAP-43, known to bind phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)]. Since palmitoylated proteins are preferentially localized in sphingolipid- and cholesterol-rich microdomains of plasma membrane, the present study has been focused on a possible effect of palmitoylcarnitine on GAP-43 localization in these microdomains. Palmitoylcarnitine treatment resulted in GAP-43 appearance in floating fractions (rafts) in sucrose gradient and increased co-localization with cholesterol and with PI(4,5)P(2), although co-localization of both lipids decreased. GAP-43 disappeared from raft fraction upon treatment with 2-bromopalmitate (an inhibitor of palmitoylating enzymes) and after treatment with etomoxir (carnitine palmitoyltransferase I inhibitor). Raft localization of GAP-43 was completely abolished by treatment with methyl-β-cyclodextrin, a cholesterol binding agent, while there was no change upon sequestration of PI(4,5)P(2) with neomycin. GAP-43 co-precipitated with a monomeric form of Gα(o), a phenomenon diminished after palmitoylcarnitine treatment and paralleled by a decrease of Gα(o) in the raft fraction. These observations point to palmitoylation of GAP-43 as a mechanism leading to an increased localization of this protein in microdomains of plasma membrane rich in cholesterol, in majority different, however, from microdomains in which PI(4,5)P(2) is present. This localization correlates with decreased interaction with Gα(o) and suppression of its activity-an important step regulating neural cell differentiation.

Dose-Dependent Expression of Neuronal Injury Markers during Experimental Osteoarthritis Induced by Monoiodoacetate in the Rat

BackgroundIt was recently reported that the mono-iodoacetate (MIA) experimental model of osteoarthritis (OA) courses with changes of neurons innervating the affected joints that are commonly interpreted as a neuronal response to axonal injury. To better characterize these changes, we evaluated the expression of two markers of neuronal damage, ATF-3 and NPY, and the growth associated protein GAP-43, in primary afferent neurons of OA animals injected with three different doses of MIA (0.3, 1 or 2 mg). Measurements were performed at days 3, 7, 14, 21 and 31 post-MIA injection.ResultsOA animals showed the characteristic histopathological changes of the joints and the accompanying nociceptive behaviour, evaluated by the Knee-Bed and CatWalk tests. An increase of ATF-3 expression was detected in the DRG of OA animals as early as 3 days after the injection of 1 or 2 mg of MIA and 7 days after the injection of 0.3 mg. NPY expression was increased in animals injected with 1 or 2 mg of MIA, at day 3 or in all time-points, respectively. From day 7 onwards there was a massive increase of GAP-43 expression in ATF-3 cells.ConclusionsThe expression of the neuronal injury markers ATF-3 and NPY as well as an up-regulation of GAP-43 expression, indicative of peripheral fibre regeneration, suggests that axonal injury and a regeneration response may be happening in this model of OA. This opens new perspectives in the unravelling of the physiopathology of the human disease.

Synaptic Reorganization in the Adult Rat's Ventral Cochlear Nucleus following Its Total Sensory Deafferentation

Ablation of a cochlea causes total sensory deafferentation of the cochlear nucleus in the brainstem, providing a model to investigate nervous degeneration and formation of new synaptic contacts in the adult brain. In a quantitative electron microscopical study on the plasticity of the central auditory system of the Wistar rat, we first determined what fraction of the total number of synaptic contact zones (SCZs) in the anteroventral cochlear nucleus (AVCN) is attributable to primary sensory innervation and how many synapses remain after total unilateral cochlear ablation. Second, we attempted to identify the potential for a deafferentation-dependent synaptogenesis. SCZs were ultrastructurally identified before and after deafferentation in tissue treated for ethanolic phosphotungstic acid (EPTA) staining. This was combined with pre-embedding immunocytochemistry for gephyrin identifying inhibitory SCZs, the growth-associated protein GAP-43, glutamate, and choline acetyltransferase. A stereological analysis of EPTA stained sections revealed 1.11±0.09 (S.E.M.)×109 SCZs per mm3 of AVCN tissue. Within 7 days of deafferentation, this number was down by 46%. Excitatory and inhibitory synapses were differentially affected on the side of deafferentation. Excitatory synapses were quickly reduced and then began to increase in number again, necessarily being complemented from sources other than cochlear neurons, while inhibitory synapses were reduced more slowly and continuously. The result was a transient rise of the relative fraction of inhibitory synapses with a decline below original levels thereafter. Synaptogenesis was inferred by the emergence of morphologically immature SCZs that were consistently associated with GAP-43 immunoreactivity. SCZs of this type were estimated to make up a fraction of close to 30% of the total synaptic population present by ten weeks after sensory deafferentation. In conclusion, there appears to be a substantial potential for network reorganization and synaptogenesis in the auditory brainstem after loss of hearing, even in the adult brain.