Csaba Szigeti

Adult rat hippocampal slices as in vitro models for neurodegeneration: Studies on cell viability and apoptotic processes

Adult hippocampal slice cultures were used in the modeling of apoptotic aspects of neurodegeneration. Slice viability was determined by the use of trypan blue (TB) staining, and apoptosis was assessed by caspase-3 immunohistochemistry. A large number of pyramidal cells showed signs of degeneration 30 min after sectioning (58.4% of the total number of pyramidal cells), as they exhibited TB uptake, and about 71.6% of these neurons became stained by the third day in culture, when patches in the stratum oriens also demonstrated distinct TB staining. By the sixth day of culturing, almost all cells in the pyramidal cell layer became TB positive (88.4%). The caspase-3 immunoreactivity displayed a different pattern, as the most intense immunoreactivity, detected mainly in the pyramidal cells, peaked 6 h after culturing, and then decreased steadily. The present data show that in adult hippocampal slices a large number of pyramidal cells initiate apoptotic processes as a result of irreparable damage sustained during slice preparation and culture maintenance, and support the notion that apoptosis is an integral part of the neurodegenerative processes not only in vivo but also in vitro. Elucidation of mechanisms for the apoptotic processes in adult hippocampal slice cultures could lead to the development of new therapeutic strategies; moreover, the utilization of adult hippocampal slice cultures could be a viable alternative technique to in vivo experiments in studying the mechanisms responsible for neurodegeneration.

Selective C-fiber deafferentation of the spinal dorsal horn prevents lesion-induced transganglionic transport of choleragenoid to the substantia gelatinosa in the rat☆

The effect of neonatal capsaicin treatment, producing selective elimination of almost all unmyelinated C-fiber sensory axons, was studied on lesion-induced transganglionic labelling of the substantia gelatinosa of the spinal cord by choleragenoid. In both control and capsaicin-pretreated rats, the injection of choleragenoid-horseradish peroxidase conjugate into the intact sciatic nerves resulted in intense labelling only of the deeper layers of the spinal dorsal horn. In the control but not the capsaicin-pretreated rats, the injection of the tracer into sciatic nerves transected 2 weeks previously produced an intense homogeneous labelling of the substantia gelatinosa. It is concluded that the uptake and axonal transport of choleragenoid by capsaicin-sensitive C-fiber afferents may be accounted for by the lesion-induced transganglionic labelling of the substantia gelatinosa, rather than by A-fiber sprouting.

Long-term effects of selective immunolesions of cholinergic neurons of the nucleus basalis magnocellularis on the ascending cholinergic pathways in the rat: A model for Alzheimer's disease

Alzheimers disease is associated with a significant decrease in the cholinergic input to the neocortex. In a rat model of this depletion, we analyzed the subsequent long-term changes in cholinergic fiber density in two well-defined areas of the frontal and parietal cortices: Fr1, the primary motor cortex, and HL, the hindlimb area of the somatosensory (parietal) cortex, two cortical cholinergic fields that receive inputs from the nucleus basalis magnocellularis (nBM). A specific cholinergic lesion was induced by the intraparenchymal injection of 192 IgG-saporin into the nBM. Choline acetyltransferase (ChAT) immunohistochemistry was applied to identify the loss of cholinergic neurons in the nBM, while acetylcholinesterase (AChE) enzyme histochemistry was used to analyze the decreases in the number of cholinoceptive neurons in the nBM and the cholinergic fiber density in the Fr1 and HL cortical areas in response to the nBM lesion. The immunotoxin differentially affected the number of ChAT- and AChE-positive neurons in the nBM. 192 IgG-saporin induced a massive, irreversible depletion of the ChAT-positive (cholinergic) neurons (to 11.7% of the control level), accompanied by a less dramatic, but similarly persistent loss of the AChE-positive (cholinoceptive) neurons (to 59.2% of the control value) in the nBM within 2 weeks after the lesion. The difference seen in the depletion of ChAT- and AChE-positive neurons is due to the specificity of the immunotoxin to cholinergic neurons. The cholinergic fiber densities in cortical areas Fr1 and HL remained similarly decreased (to 62% and 68% of the control values, respectively) up to 20 weeks. No significant rebound in AChE activity occurred either in the nBM or in the cortices during the period investigated. This study therefore demonstrated that, similarly to the very extensive reduction in the number of ChAT-positive neurons in the nBM, cortical areas Fr1 and HL underwent long-lasting reductions in the number of AChE-positive fibers in response to specific cholinergic lesioning of the nBM.

Disparate changes in the expression of transient receptor potential vanilloid type 1 receptor mRNA and protein in dorsal root ganglion neurons following local capsaicin treatment of the sciatic nerve in the rat

In situ hybridization, quantitative reverse transcription polymerase chain reaction (RT-PCR), immunohistochemistry, and Western blot analysis were applied to study the changes in expression of the major nociceptive ion channel transient receptor potential vanilloid type 1 receptor (TRPV1) after the perineural application of capsaicin or nerve transection. In control rats, quantitative morphometric and statistical analyses of TRPV1 protein and mRNA expression in L5 dorsal root ganglion cells revealed distinct populations of small (type C) and small-to-medium (type B) neurons, which showed very high and moderate levels of TRPV1, whereas larger (type A) neurons mostly did not express this receptor. After either transection or capsaicin treatment of the sciatic nerve, immunohistochemistry and Western blotting demonstrated a massive (up to 80%) decrease in the proportion of TRPV1-immunoreactive neurons and TRPV1 protein at all postoperative survival times. In situ hybridization indicated marked decreases (up to 85%) in the proportion of neurons that expressed TRPV1 mRNA after sciatic nerve transection. In contrast, although perineural treatment with capsaicin resulted in similar substantial decreases in the proportions of type B and C neurons of the L5 dorsal root ganglia 3 days postoperatively, a clear-cut tendency to recovery was observed thereafter. Hence, the proportions of both type B and C neurons expressing TRPV1 mRNA reached up to 70% of the control levels at 30 days postoperatively. In accord with these findings, quantitative RT-PCR revealed a marked and significant recovery in TRPV1 mRNA after perineural capsaicin but not after nerve transection. These observations suggest the involvement of distinct cellular mechanisms in the regulation of the TRPV1 mRNA expression of damaged neurons, specifically triggered by the nature of the injury. The present findings imply that the antinociceptive and anti-inflammatory effects of perineurally applied capsaicin involve distinct changes in neuronal TRPV1 mRNA expression and long-lasting alterations in (post)translational regulation.

Dithranol abolishes UCH-L1 immunoreactivity in the nerve fibers of the rat orofacial skin

Dithranol has been used to treat psoriasis for decades. Although its beneficial effect may involve the induction of cutaneous inflammation, and inflammation often leads to damages in nerve fibers, these alterations are not well documented. Therefore, we investigated the effects of dithranol on the immunohistochemical characteristics of the cutaneous nerve fibers in the rat skin. Epidermal nerve fiber staining was achieved with ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) immunohistochemistry in the orofacial skin of control rats, rats treated with (a) dithranol for 5 days, (b) corticosteroid for 5 days following dithranol treatment for 5 days, and (c) corticosteroid for 5 days. The results revealed a complete loss of UCH-L1 immunoreactivity in the dithranol-treated animals. Topical application of corticosteroid onto the inflamed skin for 5 days reversed this effect: the UCH-L1 immunoreactivity was almost completely restored. Steroid treatment for 5 days did not change the appearance of the UCH-L1-immunoreactive nerve fibers. These findings were supported by Western blot analyses. We conclude that dithranol, incidentally similarly to psoriasis, causes inflammation and abolishes UCH-L1 immunoreactivity in the rat orofacial skin in a corticosteroid-reversible manner. This phenomenon may be due to the ability of dithranol to cause oxidative damage to the UCH-L1 protein, and to the antioxidant activity of the corticosteroids countering this effect.

397 Effects of traumatic and selective chemical lesions of peripheral nerves on TRPV1 receptor expression: Implications for vanilloid-induced analgesia

Background. Spinal cord stimulation (SCS) is an effective tool in alleviating neuropathic pain. Previously, we have demonstrated that SCS produces an increased release of acetylcholine in the dorsal horn in SCS responding rats, and that the SCS effect involves activation of M4 and M2 muscarinic receptors. The aim of the present study was to examine whether the expression of these receptors relates to the presence of tactile hypersensitivity (‘‘allodynia’’) after partial sciatic nerve injury and to the SCS responsiveness. Methods. Tactile allodynia was assessed with von Frey filaments. A miniature electrode for SCS was implanted in the lower thoracic spinal canal. After having assessed withdrawal thresholds and the allodynia suppression effect of SCS, the lumbar spinal cord was removed and immunostained for M4 and M2 muscarinic receptors as well as for NeuN. Results. M2 receptors in the dorsal horn were unaffected by the sciatic nerve injury. In contrast, the expression of M4 receptors was significantly reduced as compared to normal rats and this effect was more prominent in allodynic than in non-allodynic rats. Further, among the allodynic rats the decreased expression was more marked in the SCS non-responding than in SCS responding ones. The reduced M4 receptor expression was not associated with a loss of neurons. Conclusions. The results indicate that M4 receptors are downregulated in spinal dorsal horn neurons following peripheral nerve injury and suggest that this effect relates to SCS responsiveness. Conversely, M2 receptors do not seem to be directly linked to the variable likeliness to respond to SCS.