Total Number Of Myelinated Fibers Research Articles

Lidocaine Nerve Block Diminishes the Effects of Therapeutic Electrical Stimulation to Enhance Nerve Regeneration in Rats.

Although electrical stimulation (ES) can improve nerve regeneration, the impact of nerve block, such as lidocaine (Lido), on the therapeutic benefits of ES remains unclear. We used a rat tibial nerve transection-and-repair model to explore how either preoperative (PreOp) or postoperative (PostOp) nerve block affects ES-related improvement in regeneration. Lewis rats were used in 1 of 2 studies. The first evaluated the effects of extraneural Lido on both healthy and injured nerves. In the second study, rats were randomized to 5 experimental groups: No ES (negative control), PreOp Lido, ES + PreOp Lido, PostOp + ES, and ES (positive control). All groups underwent tibial nerve transection and repair. In both studies, nerves were harvested for histological analysis of regeneration distal to the injury site. Application of extraneural Lido did not damage healthy or injured nerve based on qualitative histological observations. In the context of nerve transection and repair, the ES group exhibited improved axon regeneration at 21 days measured by the total number of myelinated fibers compared with No ES. Fiber density and percentage of neural tissue in the ES group were greater than those in both No ES and PreOp Lido + ES groups. ES + PostOp Lido was not different from No ES or ES group. Extraneural application of Lido did not damage nerves. Electrical stimulation augmented nerve regeneration, but Lido diminished the ES-related improvement in nerve regeneration. Clinical studies on the effects of ES to nerve regeneration may need to consider nerve block as a variable affecting ES outcome.

The total number of myelinated nerve fibers is reduced in corpus callosum in brains from patients with Alzheimer's disease.

The total number of myelinated fibers in corpus callosum (CC) in Alzheimer's disease is poorly described. Using a two-dimensional fractionator principle we estimated the total number of fibers in ten females patients with Alzheimer's disease (AD) (mean age 82.8years) and 12 female control subjects (mean age 80.6years) and found the mean number of myelinated fibers to be significantly 30% higher in the control group (90.0× 106; coefficient of variation= SD/mean= 27%) than in the AD group (69.3× 106; coefficient of variation= 20%) (p= 0.028). A significant loss of fibers (≥2.2μm in diameter) was found in the anterior part of the CC in AD patients compared to control subjects (p= 0.045). For both groups, we found a positive correlation between the total number of myelinated fibers and CC cross-sectional area (AD, r= 0.81; control subjects, r= 0.73). There was a negative correlation in both groups between age and total number of myelinated fibers (AD, r=-0.81; controls subjects, r=-0.59). These results implicate a loss of intrahemispheric connections in the pathophysiology of AD.

Long-Chain Omega-3 Fatty Acids Supplementation Accelerates Nerve Regeneration and Prevents Neuropathic Pain Behavior in Mice.

Fish oil (FO) is the main source of long chain omega-3 polyunsaturated fatty acids (ω-3 PUFAs), which display relevant analgesic and anti-inflammatory properties. Peripheral nerve injury is driven by degeneration, neuroinflammation, and neuronal plasticity which results in neuropathic pain (NP) symptoms such as allodynia and hyperalgesia. We tested the preventive effect of an EPA/DHA-concentrate fish oil (CFO) on NP development and regenerative features. Swiss mice received daily oral treatment with CFO 4.6 or 2.3 g/kg for 10 days after NP was induced by partial sciatic nerve ligation. Mechanical allodynia and thermal hypernociception were assessed 5 days after injury. CFO 2.3 g/kg significantly prevented mechanical and thermal sensitization, reduced TNF levels in the spinal cord, sciatic MPO activity, and ATF-3 expression on DRG cells. CFO improved Sciatic Functional Index (SFI) as well as electrophysiological recordings, corroborating the increased GAP43 expression and total number of myelinated fibers observed in sciatic nerve. No locomotor activity impairment was observed in CFO treated groups. These results point to the regenerative and possibly protective properties of a combined EPA and DHA oral administration after peripheral nerve injury, as well as its anti-neuroinflammatory activity, evidencing ω-3 PUFAs promising therapeutic outcomes for NP treatment.

Comparison of histopathological effects of perineural administration of bupivacaine and bupivacaine-dexmedetomidine in rat sciatic nerve

Injection of a variety of drugs such as local anesthetics (LAs) for peripheral nerve block has been shown to cause damage to peripheral nerves. Bupivacaine is a local anesthetic widely used in surgical procedures. The aim of this study was to evaluate the neurotoxicity of LAs including Bupivacaine and dexmedetomidine (DEX)-Bupivacaine on sciatic nerve tissue at histopathological level. In addition, we investigated whether perineural administration of DEX can attenuate Bupivacaine-induced neurotoxicity. Twenty adult Sprague Dawley rats received unilateral sciatic nerve blocks with either 0.2ml of 0.5% bupivacaine (n=8) or 0.5% bupivacaine plus 0.005% DEX (n=8) or normal saline (0.9%, as control group) (n=4) in the left hind extremity. Sciatic nerves were harvested at 14days post-injection and analyzed for nerve damage using ultrastructure and histopathologic analysis. Histopathology of sciatic nerve at day 14 post-injection showed a variable degree of neuronal injury associated with perineural inflammation in each treatment group and was classified as none or mild, intermediate or severe. Administration of both LAs resulted in a significant decrease in the total number of myelinated fibers per nerve (95% CI for group difference: Bupivacaine, P=0.001, DEX-Bupivacaine, P=0.036) compared to the saline control group. Animals that received these perineural local anesthetics (LAs) injections showed increased severity of injury compared to the control group. Animals in the DEX-Bupivacaine group had higher perineural inflammation and nerve damage than those of the saline control group and less than those of the Bupivacaine group at day 14 post-injection. Quantitatively, average total nerve fiber per nerve and average myelinated nerve fiber density in the injured region of the Bupivacaine-treated group was less than that of the DEX-Bupivacaine-treated group. LAs injection into the nerve causes peripheral nerve damage and remains an important clinical danger. Bupivacaine is associated with considerable histopathological changes, including edema of the perineurium and myelin degeneration with Wallerian degeneration, when injected perineurally. Perineural DEX added to a clinical concentration of bupivacaine attenuates the Bupivacaine-induced injuries.

Are we sure to count myelinated fibers entirely? Calibration of a stereological study

Peripheral nerves are subject to many different types of injuries frequently and these injuries may result in partial or total loss of motor and sensory functions. In contrast to the central nervous system, peripheral nervous system are able to undergo spontaneous regeneration. But clinical and experimental studies show that this regeneration is mostly not satisfactory. One of the most important neuropathological indicators of nerve regeneration is quantitative estimation of myelinated axons. Quantitative estimation of the total number of myelinated fibers can be obtained both by light microscopy (LM) and electron microscopy (EM) and depends on using appropriate sampling strategy. In this study, we made a direct comparison between LM and EM estimation of myelinated nerve fibers. Ten female Wistar albino rats were divided into two experimental groups; control group (n=5) and gap group (n=5). In the gap group, 5 mm right sciatic nerve segment of rats were resected to form a gap. Proximal and distal nerve stumps were surrounded by collagen membrane following resection. In the control group, rats received neither any operation nor any treatment. Rats were sacrificed at 12 week after surgery. Nerve samples were embedded in resin. Semi and ultra thin sections (of 1 μm and 100 nm thickness, respectively) were cut and stained with 1% toluidine blue (for LM) and uranyl acetate-lead citrate (for EM). Histological assessment of myelinated fibers was performed with both LM and EM using unbiased stereological counting techniques. There was a significant difference in the total number of myelinated nerve fibers between LM and EM examinations in terms of the both pure and regenerating axons. This difference is probably related to different magnification capacities in LM and EM. LM causes to underestimates as a result of possessing very small myelinated fibers, which are only detectable with EM. According to our results it may be concluded that the combination of both light and electron microscopical results is essential especially in revealing regenerating nerve fibers since very small myelinated fibers can not be distinguishable with LM.

Discrepancies in quantitative assessment of normal and regenerated peripheral nerve fibers between light and electron microscopy.

Quantitative estimation of myelinated nerve fiber number, together with fiber size parameters, is one of the most important tools for nerve regeneration research. In this study we used a design-based stereological method to evaluate the regenerative process in two experimental paradigms: crush injury and autograft repair. Samples were embedded in resin and morphometric counting and measurements were performed using both light and electron microscopes. Results show a significant difference in myelinated fiber number estimation between light and electron microscopes, especially after autograft repair; light microscope significantly underestimates the number of fibers because of the large number of very small axons that can be detected only in electron microscope. The analysis of the size parameters also shows a higher number of small fibers in electron microscopic analysis, especially in regenerated nerves. This comparative study shows that the integration of data obtained in light microscope with those obtained in electron microscope is necessary in revealing very small myelinated fibers that cannot be detected otherwise. Moreover, the difference in the estimation of total number of myelinated fibers between light and electron microscopes must be considered in data analysis to ensure accurate interpretation of the results.

Two-dimensional sample entropy analysis of rat sural nerve aging.

Entropy analysis of images are usually performed using Shannon entropy, which calculates the probability of occurrency of each gray level on the image. However, not only the pixel gray level but also the spatial distribution of pixels might be important for image analysis. On the other hand, sample entropy (SampEn) is an important tool for estimation of irregularity in time series, which calculates the probability of pattern occurrence within the series. Therefore, we propose here an extension of SampEn to a two-dimensional case, namely SampEn2D, as an entropy method for extracting features from images that accounts for the spatial distribution of pixels. SampEn2D was applied to histological segments of sural nerve obtained from young (30 days) and elderly (720 days) rats. Morphometric indexes, such as the total number of myelinated fibers and the average myelinated fibers area and perimeter were also calculated. Results show that SampEn2D can extract useful information from histological nerve images, classifying elderly rat image as more regular than young rat. As SampEn2D is related to irregularity/unpredictability, we can conclude that the proposed method is complementary to morphometric indexes. Further studies are being built to validate SampEn2D.

The human trochlear and abducens nerves at different ages - a morphometric study.

The trochlear and abducens nerves (TN and AN) control the movement of the superior oblique and lateral rectus muscles of the eyeball, respectively. Despite their immense clinical and radiological importance no morphometric data was available from a wide spectrum of age groups for comparison with either pathological or other conditions involving these nerves. In the present study, morphometry of the TN and AN was performed on twenty post-mortem samples ranging from 12-90 years of age. The nerve samples were processed for resin embedding and toluidine blue stained thin (1µm) sections were used for estimating the total number of myelinated axons by fractionator and the cross sectional area of the nerve and the axons by point counting methods. We observed that the TN was covered by a well-defined epineurium and had ill-defined fascicles, whereas the AN had multiple fascicles with scanty epineurium. Both nerves contained myelinated and unmyelinated fibers of various sizes intermingled with each other. Out of the four age groups (12-20y, 21-40y, 41-60y and >61y) the younger groups revealed isolated bundles of small thinly myelinated axons. The total number of myelinated fibers in the TN and AN at various ages ranged from 1100-3000 and 1600-7000, respectively. There was no significant change in the cross-sectional area of the nerves or the axonal area of the myelinated nerves across the age groups. However, myelin thickness increased significantly in the AN with aging (one way ANOVA). The present study provides baseline morphometric data on the human TN and AN at various ages.

Anatomical feasibility of transferring the obturator and genitofemoral nerves to repair lumbosacral plexus nerve root avulsion injuries

Nerve transfer is a valid surgical procedure for restoring lower-extremity function after lumbosacral plexus nerve root avulsion. We determined the anatomical feasibility of transferring the obturator and genitofemoral nerves for this purpose. The obturator, genitofemoral and femoral nerves, and the S1 and S2 nerve roots on both sides were exposed in 10 cadaver specimens. We traced all nerves to their origins. The lengths of the obturator and genitofemoral nerves were measured from their origins to their exits from the abdominal cavity. The transverse and longitudinal diameters of all nerves were measured. Specimens were obtained to determine the total number of myelinated fibers in each nerve. The proximal part of the left obturator nerve was anastomosed with the distal part of the right femoral nerve, between the vertebrae and the peritoneum, with an overlap of 2-3 cm. Similarly, the proximal parts of the right obturator and genitofemoral nerves were anastomosed with the ipsilateral S1 and S2 nerve roots, respectively, with an overlap of 2-4 cm. The obturator nerve contained approximately one-third of the number of fibers (4,300-7,800) presenting in the femoral nerve (13,500-21,000). Similarly, the number of fibers found in the S1 nerve root was in the range 5,200-8,900. The genitofemoral nerve contained approximately half the number of fibers (3,000-4,500) presenting in the S2 nerve root (4,600-8,400). The obturator and genitofemoral nerves could be suitable donor nerves for repairing lumbosacral plexus nerve root avulsion.

Implications of Olfactory Lamina Propria Transplantation on Hyperreflexia and Myelinated Fiber Regeneration in Rats with Complete Spinal Cord Transection

Transplantation with olfactory ensheathing cells (OECs) has been adopted after several models of spinal cord injury (SCI) with the purpose of creating a favorable environment for the re-growth of injured axons. However, a consensus on the efficacy of this cellular transplantation has yet to be reached. In order to explore alternative parameters that could demonstrate the possible restorative properties of such grafts, the present study investigated the effects of olfactory lamina propria (OLP) transplantation on hyperreflexia and myelinated fiber regeneration in adult rats with complete spinal cord transection. The efficacy of OLP (graft containing OECs) and respiratory lamina propria (RLP, graft without OECs) was tested at different post-injury times (acutely, 2- and 4-week delayed), to establish the optimum period for transplantation. In the therapeutic windows used, OLP and RLP grafts produced no considerable improvements in withdrawal reflex responses or on the low-frequency dependent depression of H-reflex. Both lamina propria grafts produced comparable results for the myelinated fiber density and for the estimated total number of myelinated fibers at the lesion site, indicating that the delayed transplantation approach does not seem to limit the regenerative effects. However, animals transplanted with OLP 2 or 4 weeks after injury exhibit smaller myelin sheath thickness and myelinated fiber area and diameter at the lesion site compared to their respective RLP groups. Despite the ongoing clinical use of OECs, it is important to emphasize the need for more experimental studies to clarify the exact nature of the repair capacity of these grafts in the treatment of SCI.

The Effect of Growth Factors and Soluble Nogo-66 Receptor Protein on Transplanted Neural Stem/Progenitor Survival and Axonal Regeneration after Complete Transection of Rat Spinal Cord

Adult central mammalian axons show minimal regeneration after spinal cord injury due to loss of oligodendrocytes, demyelination of surviving axons, absence of growth-promoting molecules, and inhibitors of axonal outgrowth. In the present study, we attempted to address these impediments to regeneration by using a combinatory strategy to enhance cell survival and regeneration after complete spinal cord transection (SCT) in adult rats. The strategy comprised: 1) adult rat brain-derived neural stem/progenitor cells (NSPCs) preseeded on laminin-coated chitosan channels; 2) extramedullary chitosan channels to promote axonal regrowth and reduce the barrier caused by scarring; 3) local delivery of a novel rat soluble Nogo-66 receptor protein [NgR(310)ecto-Fc, referred to as NgR] to block the inhibitory effect of myelin-based inhibitors; and 4) local delivery of basic fibroblast growth factor, epidermal growth factor, and platelet-derived growth factor to enhance survival and promote differentiation of transplanted cells. Compared with our previous studies where brain-derived NSPCs preseeded in extramedullary chitosan channels were implanted in the same SCT model but without growth factors and NgR, the present channel-growth factor combination produced greater numbers of surviving NSPCs after SCT. Also, the growth factors promoted preferential differentiation of NSPCs toward oligodendrocytes, while NgR significantly decreased astrocytic differentiation of NSPCs. NgR alone or in combination with NSPCs significantly enhanced the total number of myelinated fibers in the bridge and increased the area of the bridging tissue between the cord stumps. The combination of NgR, growth factors, and NSPCs had synergistic effect on bridge formation. However, only a small number of descending corticospinal tract axons grew into the central portions of the bridges as shown by anterograde tracing of the corticospinal tract with BDA. The majority of the regenerated axons in the channels originated from local host neurons adjacent to the tissue bridges. In conclusion, we showed that growth factors increased survival of transplanted NSPCs whereas NgR enhanced axonal regeneration, but the combination did not have additive effects on functional recovery or regeneration.

Short‐ and long‐term peripheral nerve regeneration using a poly‐lactic‐co‐glycolic‐acid scaffold containing nerve growth factor and glial cell line‐derived neurotrophic factor releasing microspheres

Addition of neural growth factors to bioengineered scaffolds may improve peripheral nerve regeneration. The aim of this study is to evaluate the short- and long term effect of microsphere delivered nerve growth factor (NGF) and glial cell derived neurotrophic factor (GDNF) in the 10 mm rat sciatic nerve gap. Eighty-four rats were assigned to seven groups (n = 6) at two endpoints (6 and 16 weeks): saline, saline NGF, saline NGF-microspheres, saline GDNF, saline GDNF-microspheres, saline blank microspheres, and autologous nerve graft. Total fascicular area and total number of myelinated fibers at mid-tube increased in all conduit groups between 6 and 16 weeks. Autologous, saline NGF-microsphere and saline GDNF-microsphere groups reached maximal histomorphometric values by 6 weeks (p < 0.05). Compound muscle action potentials returned after 6 weeks for the autologous graft and continued to increase to a level of 3.6 ± 1.9 mV at endpoint. No significant differences were found between study groups as measured by ankle angle. These experiments show an initial beneficial effect of incorporation of NGF- or GDNF-microspheres in a PLGA 85/15 nerve conduit, since histomorphometric values reached their maximum by 6 weeks compared to control groups. These results do not yet extrapolate into improved electrophysiological or functional improvement.

Morphometric analysis of the human superficial radial nerve

The superficial radial nerve (SRN) is one of the cutaneous nerves biopsied for diagnosis of peripheral neuropathy. Morphometric analysis is employed to provide more quantitative data regarding the histomorphology of nerve biopsy. To detect early structural changes, morphometric data from normal nerves are necessary for diagnostic criteria. Unlike the sural nerve, morphometric data on the normal SRN have not been reported. Therefore, a morphometric analysis of the normal human SRN was done in this study. The nerves were harvested bilaterally from 21 fresh human bodies, yielding 34 nerves after excluding damaged ones. Transverse semi-thin sections were analyzed for the number of fascicles, myelinated fiber and axon diameters, myelin sheath thickness, g ratio, density and total number of myelinated fibers using the three-window sampling method. We found that 2-16 fascicles were present. Mean fiber and axon diameters were 6.32 +/- 0.09 (SEM) and 3.44 +/- 0.06 microm, respectively. Average myelin thickness was 1.44 +/- 0.03 microm. Average g ratio was 0.56 +/- 0.01. Mean density of fibers was 8,872.9 +/- 167.4/mm(2). The number of fibers varied from 1,390 to 10,941, with an average of 6,495 +/- 474. These parameters were independent of age. Asymmetry in the number of fibers was also noted in four cases. The data obtained in this study can be applied to histomorphometric evaluations of SRN biopsies, although the possibility of asymmetry should be considered.

Age-related changes in human laryngeal nerves

We sought to perform a morphometric analysis of myelinated fibers from laryngeal nerves with the aim of verifying quantitative changes due to the aging process. A 1-cm fragment was collected from the superior laryngeal nerves and recurrent laryngeal nerves from 12 cadavers during autopsy. The sample was divided in two groups: an adult group (aged <60 years) and an elderly group (aged > or =60 years). The total number of myelinated fibers from the superior laryngeal nerves was similar in both groups (adult group = 9017 +/- 1692, elderly group = 7918 +/- 1624; P = 0.79). The adult group had a higher total number of myelinated fibers in the recurrent laryngeal nerves than that of the elderly group (adult group = 3276 +/- 383, elderly group = 2381 +/- 669; P = 0.005), as well as a higher total number of fibers in the laryngeal nerves (both superior and recurrent) than the elderly group (P = 0.02). The adult group has a higher total number of myelinated fibers in the laryngeal nerves than the elderly group.

Repair of peripheral nerve defect with direct gradual lengthening of the proximal nerve stump in rats

We investigated the effect of direct gradual lengthening on the proximal nerve stump and subsequent nerve regeneration in rats. A 10-mm-long nerve segment was resected from the sciatic nerve of each rat. The proximal nerve stump was directly lengthened at a rate of 1 mm/day using an original external nerve distraction device. Experiment I: After distraction periods of 10, 15, and 20 days, the length of each nerve was evaluated, and the lengthened nerve stump was also examined by immunohistochemical analysis. Experiment II: After a distraction period of 20 days, both nerve stumps were refreshed and direct end-to-end neurorrhaphy was performed. For control, 10-mm nerve grafting was immediately performed after nerve resection. Nerve regeneration was evaluated electrophysiologically and histologically 7, 9, and 15 weeks after nerve resection in both groups. The whole proximal nerve stump, including the endoneurium and the axon, could be lengthened in proportion to the distraction period. There were no significant differences in motor nerve conduction velocity and tetanic muscle contraction force between both groups. Histologically, the total number of myelinated fibers was significantly greater in the nerve lengthening group than in the autografting group. This study demonstrated that the whole proximal nerve stump including the endoneurium and the axon could be lengthened by direct gradual distraction, and that this method might have potential application in the repair of peripheral nerve defects.

Ro5-4864, a synthetic ligand of peripheral benzodiazepine receptor, reduces aging-associated myelin degeneration in the sciatic nerve of male rats

The peripheral-type benzodiazepine receptor (PBR) is a protein predominantly located in the mitochondrial outer membrane that plays an important role in the regulation of cell survival and proliferation. Previous studies have shown an enhanced expression of PBR in the regenerating sciatic nerve, suggesting that this protein may be involved in the regenerative response. The rat sciatic nerve suffers important structural alterations with aging, including alterations in the morphology of myelin sheaths and a decrease in the number of myelinated fibers. In this study, we have assessed the effect of two PBR ligands, Ro5-4864 and PK-11195, to determine whether PBR may influence aging-associated morphological changes in the sciatic nerve. The treatment of 23-month-old, Sprague–Dawley male rats for 1 month with Ro5-4864 significantly reduced the percentage of fibers with myelin decompaction and increased the total number of myelinated fibers. In contrast, PK-11195, a PBR ligand that binds to a different site than Ro5-4864 in the PBR molecule, did not significantly affect any of the parameters analyzed. These findings support the potential role of PBR ligands to prevent aging-associated peripheral nerve degeneration.

Accuracy of sampling methods in morphometric studies of human sural nerves

The aim of this study was to ascertain the minimum sample required to accurately measure the total number of myelinated fibres, mean myelinated fibre density (MFD), myelinated fibre diameter (Ds) and axonal diameter (Da) in morphometric studies of sural nerve biopsies. Measurements were obtained by sampling a single fascicle or systematic sampling of up to 50% of the total transverse fascicular area of two control and eighteen pathological sural nerves showing varying degrees of demyelination and axonal degeneration. MFD and fibre size were heterogeneous between fascicles in both control and pathological sural nerves, and morphometric results from one fascicle and systematic sampling of up to 50% of the total transverse fascicular area did not accurately represent the whole myelinated fibre population in the sural nerve. For accurate morphometric data it is necessary to quantitate all the myelinated fibres in the sural nerve.

Dehydroepiandrosterone as an enhancer of functional recovery following crush injury to rat sciatic nerve.

This study was designed to investigate the effect of dehydroepiandrosterone (DHEA) on the recovery of the rat sciatic nerve following crush injury. A standard hemostat system was used to create the injury, with a length of 1.5 mm in three groups of 18 animals each. In group I, the crush injury was applied without any treatment. In groups II and III, vehicle (ethylene glycol) and DHEA solutions were injected subepineurally 30 min following the crush injury. Sciatic function index (SFI), toe contracture measurement, gastrocinemius muscle weight, total number of myelinated fibers, fiber diameters, myelin thickness, and axon/fiber cross-sectional ratio were measured at 3, 6, and 12 weeks. The SFI values in the DHEA group showed a faster return to normal values confirmed at 3 and 6 weeks (P < 0.05). The number of myelinated fibers and fiber diameters at 6 and 12 weeks were significantly higher in the DHEA group (P < 0.05). In this study, the subepineural injection of DHEA following crush injury was found to enhance functional recovery of the rat sciatic nerve.

Verification of the two-dimensional disector, a method for the unbiased estimation of density and number of myelinated nerve fibers in peripheral nerves

Quantification of the number of myelinated fibers in peripheral nerves is a common requirement in quantitative morphology. This parameter provides important information on the consequences of various physiological, pathological and experimental conditions on the nerve structure and is one of the main indicators of success of peripheral nerve repair. In this paper, the theoretical rationale for the application of stereological principles to obtain unbiased estimates of the density and total number of myelinated fibers in peripheral nerves is discussed and a simple stereological method is described. The method is applied together with a systematic random sampling scheme, that was optimized for the purposes of the present study, and with sampling scheme analysis by calculating the coefficient of error (CE). The stereological method, which consists of a two-dimensional variation of the classical disector procedure (two-dimensional disector), and the sampling scheme are verified by comparing estimates with the true density and total number of myelinated fibers in peripheral nerve trunks where true values have been accurately determined by extensive counting. The verification of the 2-D disector method, both of normal and regenerated nerves, showed that estimates of density and total number of myelinated nerve fibers are unbiased. The method also proved to be efficient (time-saving): Estimation of density and total number of myelinated fibers in a single nerve takes about 2-3 hours.

Fibre composition in the interosseous nerve of the pigeon.

The interosseous nerve of birds innervates a string of Herbst corpuscles located near the interosseous membrane between the tibia and fibula. Fibre composition of this nerve was assessed including both myelinated and unmyelinated axons. The diameter of the whole nerve is approximately 100 microm. Complete data were obtained for 3 nerves. The mean total number of myelinated fibres and unmyelinated axons was 2872 +/- 53. The mean number of myelinated fibres was 280 +/- 20 and that for unmyelinated axons was 2600 +/- 47. There was a broad distribution of diameters for myelinated fibres ranging from approximately 2 microm to 10 microm with a distinct peak at approximately 3-5 microm and a less prominent second peak at 6-8 microm. Similarly, myelin sheath thickness distribution showed 2 peaks, one at 0.6-0.8 microm and another at 1.4-1.6 microm. It is suggested that the group represented by the second peak innervates the Herbst corpuscles. The group of smaller myelinated fibres and the unmyelinated axons are assumed to innervate other types of receptors, some of which may be nociceptors.