The toxic effect of non-steroidal anti-inflammatory drugs (NSAIDs) during development has been widely investigated. While it has been shown that these drugs impair central nervous development and compromise the neural activity, the effects of these substances on the development of peripheral nerves are still not clarified. In the present study, sciatic nerves withdrawn from three experimental groups of 4-week-old rats, prenatally exposed to either saline solution, or diclofenac sodium, and controls not exposed to any substance, were evaluated in terms of axon number, cross-sectional area of axon and myelin sheet thickness as well as of the ultrastructure of nerve fibers. Comparisons of stereological estimations among these three groups showed that axon number and mean axon cross-sectional area, but not average myelin sheet thickness, were significantly decreased in rats that were exposed to both diclofenac sodium and also to the saline solution, in comparison of the control group. Electron microscope analysis revealed, in both treated groups, deterioration of myelin sheaths that was more pronounced in rats that were exposed to diclofenac sodium. Altogether, these findings show that the prenatal administration of both diclofenac sodium and saline solution impairs peripheral nervous system development, thus suggesting that this potential teratogenic effect should be also taken into consideration in the clinical use of these substances in pregnant patients.

Several sources of variability can affect stereological estimates. Here we measured the impact of potential sources of variability on numerical stereological estimates of myelinated axons in the adult rat sciatic nerve. Besides biological variation, parameters tested included two variations of stereological methods (unbiased counting frame versus 2D-disector), two sampling schemes (few large versus frequent small sampling boxes), and workstations with varying degrees of sophistication. All estimates were validated against exhaustive counts of the same nerve cross sections to obtain calibrated true numbers of myelinated axons (gold standard). In addition, we quantified errors in particle identification by comparing light microscopic and electron microscopic images of selected consecutive sections. Biological variation was 15.6%. There was no significant difference between the two stereological approaches or workstations used, but sampling schemes with few large samples yielded larger differences (20.7%±3.7% SEM) of estimates from true values, while frequent small samples showed significantly smaller differences (12.7%±1.9% SEM). Particle identification was accurate in 94% of cases (range: 89-98%). The most common identification error was due to profiles of Schwann cell nuclei mimicking profiles of small myelinated nerve fibers. We recommend sampling frequent small rather than few large areas, and conclude that workstations with basic stereological equipment are sufficient to obtain accurate estimates. Electron microscopic verification showed that particle misidentification had a surprisingly variable and large impact of up to 11%, corresponding to 2/3 of the biological variation (15.6%). Thus, errors in particle identification require further attention, and we provide a simple nerve fiber recognition test to assist investigators with self-testing and training.

Nerve regeneration after surgical reconstruction is far from optimal, and thus effective strategies for improving the outcome of nerve repair are being sought. In this experiment, we verified if postoperative intraperitoneal melatonin (MLT) administration after intraoperative platelet gel application improves peripheral nerve regeneration. In adult male rats, 1-cm long sciatic nerve defects were repaired using four different strategies: autologous nerve graft repair followed by MLT (NM, n = 5), collagen conduit repair followed by MLT (CM, n = 5), platelet gel-enriched collagen conduit repair followed by MLT (CGM, n = 6), and platelet gel-enriched collagen conduit (CG, n = 5) repair followed by no substance administration. Sham operated animals were used as controls (Cont, n = 5). Ninety days after surgery, the nerve regeneration outcome was comparatively assessed by means of electrophysiological and stereological analysis. Electrophysiology revealed no significant differences between the experimental and the sham control groups. Stereological analysis showed no significant differences among the experimental groups regarding axon size and myelin thickness, but the axon number was significantly lower in the CM compared to Cont and NM group. Moreover, there was no significant difference between number of axons in CG and Cont groups, between CGM and CM, and between CM and NM. Although it was observed that platelet gel have a positive effect on nerve regeneration, but a combination of local platelet gel with MLT does not have the same effect on nerve repair.

The potential ability of melatonin to protect against impairment of the fetal peripheral nerve system due to maternal consumption of diclofenac sodium (DS) was investigated. Eighty-four pregnant rats were divided into seven groups: control (CONT), saline administered (PS), DS administered (DS), DS with low-dose melatonin administered (DS+MLT10), DS with high-dose melatonin administered (DS+MLT50), low-dose melatonin administered (MLT10), and high-dose melatonin administered (MLT50). After the pregnancy, six male newborn rats from each group were sacrificed at 4 and 20 weeks of age. Their right sciatic nerves were harvested, and nerve fibers were evaluated using stereological techniques. Mean numbers of myelinated axons, axon cross-section areas and the mean thickness of the myelin sheet were estimated. Four-week-old prenatally DS-exposed rats had significantly fewer axons, a smaller myelinated axonal area, and a thinner myelin sheath compared to CONT group (p<0.05). Although melatonin at both doses significantly increased axon numbers, only a high dose of melatonin increased the diameter of those axons (p<0.05). At 20-weeks of age, myelinated axon number in the DS group was not only significantly lower than all other groups (p<0.05) but also the cross-sectional area of these axons was smaller than all other groups (p<0.05). There were no differences between the groups regarding the mean thickness of the myelin sheet. The current study indicates that prenatal exposure to DS decreases the number and the diameter of sciatic nerve axons and that melatonin prophylaxis can prevent these effects.

The aim of this study was to investigate the role of hydroxychloroquine (HCQ)-induced oxidative stress on sciatic nerve and muscle tissues of rats. The oxidant/antioxidant parameters in the sciatic nerve and muscle tissues were analyzed, and stereological analysis of the sciatic nerve was performed. Levels of malondialdehyde and nitric oxide in the tissues were significantly higher in the HCQ group than in the control group (p < 0.05). In addition, activities of superoxide dismutase and glutathione peroxidase were found to be significantly higher in the HCQ group than the control group (p < 0.05). There were significant decreases in nerve fiber diameter and myelin sheet thickness in the HCQ group compared with the control group (p < 0.05). These results revealed that HCQ might increase oxidative stress on sciatic nerve and muscle tissues of rats, which may correlate with axonal atrophy in sciatic nerves.

Histological and stereological results indicated that treatment with DS or saline produced undesirable effects on female rat optic nerve development and myelinization with respect to morphology.

There has been no study aimed at directly determining of the periods of Sertoli cell proliferation in birds even domestic fowl. The aims of this study were to observe the cessation of post-hatching mitotic proliferation of Sertoli cells in domestic fowl, and to determine the volume density of Sertoli and germ cells during this period. A total of 50 Leghorn chicks were used in this study. The testes sections of the animals were immunostained with BrdU to observe the proliferation of cells from one to 10 weeks of age. The volume density of the Sertoli and germ cells were determined using the standard point counting method. The volume density of the germ cell nuclei was initially less than that of the Sertoli cells but the volume density converged by week 6, and remained relatively constant until the commencement of meiosis. Clear labeling of Sertoli and germ cells was observed from week 1 to week 7. The only those cells still labeled after 8 weeks were germ cells, indicating that Sertoli cell proliferation had ceased. Therefore, it is recommended that any research into the testes of domestic fowl should consider the cessation of Sertoli cell proliferation by approximately 8 weeks.

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