B Golka

Regeneration of sciatic nerves of adult rats induced by extracts from distal stumps of pre-degenerated peripheral nerves

Despite numerous experimental and clinical attempts to reconstruct injuries of peripheral nerves, the methods developed until now have not been sufficiently effective. We examined the influence of extracts (postmicrosomal fractions) obtained from non‐pre‐degenerated or 7‐day‐pre‐degenerated distal segments of peripheral nerves on the regeneration of injured sciatic nerves of male adult rats. The extracts were introduced to the site of injury with autologous connective tissue chambers filled with fibrin. Reference groups were treated with brain‐derived neurotrophic factor (BDNF) or fibrin only. We examined DiI‐labeled motoneurons, toluidine blue‐labeled myelinated fibers in the mid‐part of the chambers, and AChE‐positive nerve endings to assess the regeneration intensity. In addition, the length of fibers regrowing within the chambers was measured. We found that extracts obtained from distal stumps of 7‐day‐pre‐degenerated peripheral nerves enhanced nerve regeneration as strongly as BDNF.

Predegenerated Peripheral Nerve Grafts Rescue Retinal Ganglion Cells from Axotomy-Induced Death

The inability of axons to grow across damaged central nervous system tissue is a well-known consequence of injury to the brain and spinal cord of adult mammals. Our previous studies showed that predegenerated peripheral nerve grafts facilitate neurite outgrowth from the injured hippocampus and that this effect was particularly distinct when 7-, 28-, and 35-day-predegenerated nerve grafts were used. The purpose of the present study was to use the above method to induce and support the regrowth of injured nerve fibers as well as the survival of retinal ganglion cells (RGCs). Adult Sprague-Dawley rats were assigned to three groups. In the experimental groups transected optic nerve was grafted with peripheral nerve (predegenerated for 7 days (PD) or nonpredegenerated). In the control group, the optic nerve was totally transected. RGCs and growing fibers labeled with fluorescent tracers were examined. They were counted and the results were subjected to statistical analysis. Retinal ganglion cells survived in the groups treated with predegenerated as well as nonpredegenerated grafts; however, the number of surviving retinal ganglion cells was significantly higher in the first one. In both groups the regrowth of the transected optic nerve was observed but the distance covered by regenerating fibers was longer in the PD group. No fibers inside grafts and no labeled cells in retinas were present in the control animals. On the basis of the obtained results we can state that the predegeneration of grafts enhance their neurotrophic influence upon the injured retinal ganglion cells.

Neurotrophic effect of submicrosomal fractions obtained from pre-degenerated peripheral nerves

Submicrosomal fractions obtained from pre-degenerated distal stumps of sciatic nerves were implanted by means of connective tissue chambers into the injured hippocampus for 8 and 18 weeks. The nerve stumps were allowed to pre-degenerate for 7, 28 and 35 days. The neuronal outgrowth was examined by means of FITC-HRP injected into the chamber. Eight weeks postoperatively the greatest number of traced cells was present in brains treated with the fraction obtained from nerves pre-degenerated for 7 days. Eighteen weeks following implantation the greatest number of FITC-HRP positive cells was found in brains grafted with the fraction from nerves pre-degenerated for 35 days.

Neurotrophic activity of extracts from distal stumps of pre-degenerated peripheral rat nerves varies according to molecular mass spectrum

Abstract Objective: We investigated neurotrophic activity of extracts from pre-degenerated and non-pre-degenerated peripheral nerves (complete extracts and extracts with fractions of narrower range of molecular weight) on the injured hippocampus. Methods: The experiment was carried out on male Wistar C rats. The complete extracts or fractions with different ranges of molecular weight were introduced to the site of injury with the autologous connective tissue chambers. We examined DiI-labeled hippocampal cell and AChE-positive nerve endings to assess the regeneration intensity. Results: The highest number of labeled hippocampal cells was observed in the group treated with fraction of molecular weight 10–100 kDa (72.5 ± 13.7) obtained from pre-degenerated nerves. We observed the presence of AChE-positive fibers inside all examined chambers. Discussion: These results demonstrate that suitable modification of CNS environments by introducing the protein fractions obtained from peripheral nerves can initiate the regeneration of the damaged hippocampal structure in adult rats. Moreover, it is possible to intensify their neurotrophic effect by former pre-degeneration of peripheral nerves and extraction from the entire extract proteins of molecular weight of 10–100 kDa.

The changes in neurotrophic properties of the peripheral nerves extracts following blocking of BDNF activity

Abstract Objectives: Retinal ganglion cells (RGCs) of adult rats are unable to regenerate their axons after optic nerve injury and soon after they enter the pathway of apoptosis. They may, however, survive and regenerate new axons in response to application of specific peripheral nerve extracts that presumably contain a range of neurotrophic substances. One of the recognized substances of proven neurotrophic activity is brain-derived neurotrophic factor (BDNF). We have investigated whether blocking the BDNF activity in post-microsomal fractions obtained from 7 day pre-degenerated peripheral nerves would affect its neurotrophic properties towards RGCs after optic nerve transection in adult rats. Methods: Autologous connective tissue chambers sutured to the distal end of transected optic nerve served as active substances containers. Surviving RGCs were visualized using Dil. The number of myelinated outgrowing fibers within the chambers was evaluated in histologic sections. Results: BDNF and 7 day pre-degenerated nerve extracts, and also extracts with blocked BDNF activity, enhanced RGC fibers outgrowth. The regeneration was significantly weaker in the control group. Blocking the BDNF activity in the 7 day pre-degenerated peripheral nerve extract reduced its neurotrophic effects but the differences were insignificant in comparison with non-blocked extracts. Discussion: The regeneration intensities in groups receiving 7 day pre-degenerated peripheral nerve extracts (PD7) and BDNF were comparable. The number of surviving cells was higher in the PD7 group and there were more regenerating fibers in the BDNF group, which may be explained by the strong BDNF effect on axonal collateralization and sprouting.

Experimental hyperthyroidism enhances the regeneration of central neurites promoted by peripheral nerve grafts in the hippocampus.

The aim of the present paper was to ascertain whether experimental hyperthyroidism promotes the regenerative action of predegenerated peripheral nerve grafts implanted into the transected hippocampus. Hyperthyroidism was induced by subcutaneous injections of T4. Autologous peripheral nerve grafts were implanted immediately, 7 and 35 days following transection of the sciatic nerve. Cells extending their neurites into the grafts were traced by means of horseradish peroxidase conjugated with fluoresceine isothiocyanate (FITC-HRP). Fluorescence microscope examination revealed that experimentally induced hyperthyroidism considerably enhanced the regenerative influence of peripheral nerve grafts. This effect was particularly pronounced in hyperthyrotic animals treated with either nonpredegenerated or 35 day predegenerated nerve grafts.