Viability of nerve grafts preserved in different storage medium: ultrastructural features

We investigated the ultrastructural organization of transplanted autologous grafts after storage in two different solutions. Male Wistar rats were divided into groups to obtain normal tibial nerves, freshly transplanted nerves, and nerves stored in Wisconsin/Belzer or Collins solution for 24 or 72 hours at 4 °C and transplanted (W1, W3, C1, C3). After storage or transplantation, the specimens were processed for ultrastructural analysis. All grafts showed alterations in collagen fiber organization in the endoneurial space compared to normal nerves. These fibers were more loosely organized among nerve fibers, a finding that was significantly more marked in group C3 compared to groups W1 and W3. Important alterations were also observed in the myelin sheath structure of grafts stored in the two media. These changes were characterized by separation of the lipid lamellae, clearly visible in larger diameter nerve fibers. These findings were more marked and frequent in the C1 and C3 groups compared to the W1 and W3 groups. Ultrastructural analysis showed better preservation of Schwann cells and other elements that support axonal regeneration for grafts stored in Wisconsin/Belzer solution. These results support ongoing studies for the formulation of storage solutions that permit the creation of nerve banks for heterologous transplantation.

Effects of systemic administration of ciliary neurotrophic factor on Bax and Bcl-2 proteins in the lumbar spinal cord of neonatal rats after sciatic nerve transection

Ciliary neurotrophic factor (CNTF) is a cytokine that plays a neuroprotective role in relation to axotomized motoneurons. We determined the effect of daily subcutaneous doses of CNTF (1.2 µg/g for 5 days; N = 13) or PBS (N = 13) on the levels of mRNA for Bcl-2 and Bax, as well as the expression and inter-association of Bcl-2 and Bax proteins, and the survival of motoneurons in the spinal cord lumbar enlargement of 2-day-old Wistar rats after sciatic nerve transection. Five days after transection, the effects were evaluated on histological and molecular levels using Nissl staining, immunoprecipitation, Western blot analysis, and reverse transcriptase-polymerase chain reaction. The motoneuron survival ratio, defined as the ratio between the number of motoneurons counted on the lesioned side vs those on the unlesioned side, was calculated. This ratio was 0.77 ± 0.02 for CNTF-treated rats vs 0.53 ± 0.02 for the PBS-treated controls (P < 0.001). Treatment with CNTF modified the level of mRNA, with the expression of Bax RNA decreasing 18 percent (with a consequent decrease in the level of Bax protein), while the expression of Bcl-2 RNA was increased 87 percent, although the level of Bcl-2 protein was unchanged. The amount of Bcl-2/Bax heterodimer increased 91 percent over that found in the PBS-treated controls. These data show, for the first time, that the neuroprotective effect of CNTF on neonatal rat axotomized motoneurons is associated with a reduction in free Bax, due to the inhibition of Bax expression, as well as increased Bcl-2/Bax heterodimerization. Thus, the neuroprotective action of the CNTF on axotomized motoneurons can be related to the inhibition of this apoptotic pathway.

Non-neuronal cells are not the limiting factor for the low axonal regeneration in C57BL/6J mice

Peripheral axonal regeneration was investigated in adult male mice of the C57BL/6J (C), BALB/cJ (B) and A/J (A) strains and in their F1 descendants using a predegenerated nerve transplantation model. Four types of transplants were performed: 1) isotransplants between animals of the C, B and A strains; 2) donors of the C strain and recipients of the C x B and C x A breeding; 3) donors of the B strain and recipients of the C x B breeding, and 4) donors of the A strain and recipients of the C x A breeding. Donors had the left sciatic nerve transected and two weeks later a segment of the distal stump was transplanted into the recipient. Four weeks after transplantation the regenerated nerves were used to determine the total number of regenerated myelinated fibers (TMF), diameter of myelinated fibers (FD) and myelin thickness (MT). The highest TMF values were obtained in the groups where C57BL/6J mice were the donors (C to F1 (C x B) = 4658 + OR - 304; C to F1 (C x A) = 3899 + OR - 198). Also, A/J grafts led to a significantly higher TMF (A to F1 (C x A) = 3933 + OR - 565). Additionally, isotransplant experiments showed that when the nerve is previously degenerated, C57BL/6J mice display the largest number of myelinated fibers (C to C = 3136 + OR - 287; B to B = 2759 + OR - 170, and A to A = 2835 + OR - 239). We also observed that when C57BL/6J was the graft donor, FD was the highest and MT did not differ significantly when compared with the other groups. These morphometric results reinforce the idea that Schwann cells and the nerve environment of C57BL/6J provide enough support to the regenerative process. In this respect, the present results support the hypothesis that the non-neuronal cells, mainly Schwann cells, present in the sciatic nerve of C57BL/6J mice are not the main limiting factor responsible for low axonal regeneration

Exogenous ganglioside stimulation of axonal regeneration after new transection and entubulation repair

Adult male mice received sciatic nerve transection at the midthigh level and both nerve stumps were sutured into a polythylene tube (PT) to bridge a nerve gap of 4 mm. The tubes were implanted either empty, or filled with collagen alone or in combination with gangliosides (GM1, GD1a, GD1b, anhd GT1b). Following a survival time of 6 weeks, the PT with the regenerating nerve cables were processed for plastic embedding, and morphometric measurements were made on myelinated and unmyelinated axons. The data suggest that local application of exogenous gangliosides causes a stimulation of axonal sprouting in vivo with no effefct on the rate of axonal maturation

Addition of nerve growth factor to the interior of a tubular prosthesis increases sensory neuron regeneration in vivo

The sciatic nerve of adult mice was transected and the proximal and distal nerve stumps were sutured into a polyethylene tube. The tubes were implanted either umpty, or the lumen was filled with pure collagen or a mixture of collagen/nerve trowth factor (NGF). Six weeks later, cells in the L3-L5 dorsal root ganglia (DRG) were retrogradely filled with horseradish perocidase (HRP). The data demonstrate that the addition of NGF to the interior of the tubular prosthesis can significantly increase the regeneration rate of sensory neurons