Trine Tandrup

Delayed loss of small dorsal root ganglion cells after transection of the rat sciatic nerve.

The present study deals with changes in numbers and sizes of primary afferent neurons (dorsal root ganglion [DRG] cells) after sciatic nerve transection. We find that this lesion in adult rats leads to death of some DRG cells by 8 weeks and 37% by 32 weeks after the lesion. The loss of cells appears earlier in and is more severe in B‐cells (small, dark cells with unmyelinated axons) than A‐cells (large, light cells with myelinated axons). With regard to mean cell volumes, there is a tendency for both categories of DRG cells to be smaller, but except for isolated time points, these differences are not statistically significant. These findings differ from most earlier reports in that the cell loss takes place later than usually reported, that the loss is more severe for B‐cells, and that neither A‐ or B‐cells change size significantly. Accordingly, we conclude that sciatic nerve transection in adult rats leads to a slowly developing but relatively profound loss of primary afferent neurons that is more severe for B‐cells. These results can serve as a basis for studies to determine the effectiveness of trophic or survival factors in avoiding axotomy induced cell death. J. Comp. Neurol. 422:172–180, 2000.

Effect of permanent axotomy on number and volume of dorsal root ganglion cell bodies

The effects of axotomy on the size and number of rat dorsal root ganglion cells was studied using stereological methods. Twenty adult Wistar rats were axotomized by transection of the right fifth lumbar spinal nerve approximately 7 mm distal to the fifth lumbar dorsal root ganglion (DRG‐L5). The corresponding ganglia from the nonaxotomized side served as controls. The DRG‐L5 were removed for study 4, 8, 15, and 45 days after axotomy. The number of neurons in each DRG‐L5 was determined from estimates of the numerical density, NV, made with disectors and estimates of the volume of the ganglion using the Cavalieri principle. The mean cell body volume was determined with the vertical planar rotator method. There was a progressive loss of nerve cells during the postoperative period. There was a loss of 6% (not significant) after 4 days, 19% (not significant) after 8 days, 22% (2P < 0.05) after 15 days, and 35% (2P < 0.005) after 45 days. The relative reduction in cell number 45 days after axotomy was larger for B‐cells (43%) than for A‐cells (15%). The mean nerve cell body volume for the entire DRG‐L5 cell population was reduced by 33% (2P < 0.005) 4 days after axotomy and remained so throughout the experimental period. The distribution of the individual cell volumes in the ganglia appeared to be uniformly shifted to lower values. It is concluded that permanent axotomy of the fifth lumbar spinal nerve results in a substantial loss of dorsal root ganglion cells and is well‐suited as a model for studying the potential protective effects of neurotrophic factors using modern stereological techniques. J. Comp. Neurol. 388:307–312, 1997.

Unbiased estimates of number and size of rat dorsal root ganglion cells in studies of structure and cell survival.

For quantitative studies of rat dorsal root ganglion (DRG) in experimental models stereological principles offer a number of different techniques. The application, however, requires knowledge of the anatomy and cytology of the ganglion, considerations of sampling and choosing between the many estimators available. For number and volume estimates in thick glycolmethacrylate sections the optical fractionator and the vertical planar rotator technique in most cases provide sufficient efficiency and are simple to use. Classification of the neurons in the DRG into A- and B-cells is of value in experimental conditions where the two cell types can react differently. Studies on development and subclassification of neuronal DRG cells will gain from application of stereological methods, also. Until now the methods have mainly been applied in studies of axotomy and in a few intoxication models where the time course of cell loss and changes in perikarya volume are important parameters. Further quantitative studies providing better understanding of distribution and expression of neuropeptides, cytokines, apoptotic molecules etc. will provide insight for future therapeutic approaches in neurodegenerative disorders. The more demanding staining techniques require less restrictive embedding media, but unbiased principles are available for almost all the stereological techniques applied to tissue only deformed after sectioning.

Effect of nerve crush on perikaryal number and volume of neurons in adult rat dorsal root ganglion

Assumption‐free stereological methods were applied to assess the effect of nerve crush on perikaryal number and mean volume of neuronal subpopulations in adult rat dorsal root ganglion (DRG). The L5 spinal nerve of 20 Wistar rats was crushed approximately 7 mm distal to the DRG, and the contralateral spinal nerve and DRG were left intact and used as controls. After four, 15, 45, and 120 days, the rats were killed, and the tissue was fixed and processed for subsequent preparation of 30‐μm‐thick sections. Estimates of neuron number were obtained with the optical fractionator technique and estimates of the mean perikaryal volume with the vertical planar rotator principle. Perikaryal loss was progressive during the early study period but stabilized 45 days after nerve injury. The mean number (n) of all neurons in intact L5 DRG was 16,400 (S.D. = 2,000). The loss of perikarya was 16% (P < 0.05) after four days, 15% (P < 0.05) after 15 days, 30% (P = 0.059) after 45 days, and 34% (P < 0.05) after 120 days. B cells were lost at an earlier time than were A cells, and the B cell loss was more pronounced (39% vs. 22%, respectively, after 120 days). For A cells, the mean perikaryal volume was initially reduced but was normalized at the end of the study. Distributions of perikaryal volume showed that the curves of both A and B cells were uniformly displaced toward smaller values 15 and 45 days after injury. Neuronal loss caused by crush seems similar to that seen in rats exposed to permanent axotomy (Vestergaard et al. [1997] J Comp Neurol 388:307–312) at the same location, indicating that survival of perikarya is not dependent on possibility for fiber growth. J. Comp. Neurol. 412:186–192, 1999.

Selective degeneration of dorsal root ganglia and dorsal nerve roots in methyl mercury-intoxicated rats: a stereological study

Abstract The components of the nervous system of rats that are most critically affected by methyl mercury are still a matter of debate. A recent stereological study of rats with typical symptoms resulting from methyl mercury intoxication demonstrated that the morphology of cerebellar granule cells and Purkinje cells were unchanged at the light microscopic level, even though there was pronounced degeneration of myelinated axons in dorsal nerve root nerves. In the present study, unbiased stereological methods were used to quantify morphological changes in the dorsal root ganglion, and dorsal and ventral nerve roots of the rats used in the previous study. The rats were treated with methyl mercury (2 mg daily/kg, per os) for a 19-day period that was followed by a 32-day period without treatment. The means of the total numbers of A-cell and B-cell perikarya in the dorsal root ganglion of the intoxicated rats were reduced by 60% and 24%, respectively. The mean volume of A-cell perikarya in rats of the experimental group was reduced by 22%, whereas the mean volume of B-cell perikarya was the same in the two groups. In the experimental group, the total number of myelinated axons in the dorsal nerve roots was reduced by 60%, whereas no difference was found in the ventral nerve roots. The areas of axon and myelin sheath, dorsal and ventral nerve roots were not affected. This study demonstrates that extensive loss of dorsal root ganglion cells and myelinated axons in dorsal nerve roots precedes light microscopical changes in the ventral nerve roots and the cerebellum of rats intoxicated with methyl mercury.

NUMBER AND VOLUME OF RAT DORSAL ROOT GANGLION CELLS IN ACRYLAMIDE INTOXICATION

SummaryAcrylamide intoxication induces a filamentous neuropathy with breakdown of distal axons and chromatolytic reaction of dorsal root ganglion cells. To obtain quantitative information about the perikaryal alterations neurons of the fifth lumbar dorsal root ganglion of rats were examined with stereological techniques following intoxication with a total dose of 500 mg acrylamide. Number, mean volume and distribution of neuron volume were estimated for each of the two cell subpopulations using optical disectors, the four-way-nucleator and systematic sampling techniques. In intoxicated rats perikaryal volume of A-cells was significantly reduced by 28%, from 63 200 μm3 (CV=0.16) to 45 500 μm3 (CV=0.19), whereas the volume of B-cells was unchanged. Numbers of A- and B-cells were preserved. The finding of a selective atrophy of A-cell perikaryal volume is in accordance with previous observations of predominant alterations of large myelinated sensory fibres and most likely reflects an attack on the perikaryal neurofilaments abundant in this cell type.

No further loss of dorsal root ganglion cells after axotomy in p75 neurotrophin receptor knockout mice

The role of the p75 neurotrophin receptor for neuronal survival after nerve crush was studied in L5 dorsal root ganglia (DRG) of knockout mice and controls with assumption‐free stereological methods. Numbers of neuronal A‐ and B‐cells were obtained using the optical fractionator and optical disector techniques. At birth, the total number of DRG neurons was 10,000 ± 2,600 in control mice compared with 5,100 ± 1,300 in p75 knockout mice. During postnatal development, 1,400 neuronal B‐cell bodies were lost in p75 knockouts (2P < 0.05) and 1,100 in controls (NS), whereas the A‐cell population remained stable. After a sciatic nerve crush, the total neuron loss in controls was 15.4% ± 3.5% (2P < 0.05) and 22.7% ± 5.1% (2P < 0.05) at days 14 and 42, respectively. In contrast, there was no loss in total number of neurons after crush in p75 knockout mice. Neuronal A‐cell number was unchanged after the crush in p75 knockouts as well as in controls at both times. At 14 days, the population of B‐cells was reduced by 24.8% ± 3.6% in controls and by 6.1% ± 3.5% in p75 knockouts, this difference being significant (2P < 0.001). At 42 days, the B‐cell loss was 29.6% ± 5.5% in controls and 4.2% ± 6.4% in p75 knockouts (2P < 0.001). In conclusion, the lack of the p75 receptor results in neuronal DRG cells that are resistant to nerve injury, pointing to a role for the receptor in apoptosis. J. Comp. Neurol. 459:242–250, 2003.

The volume of Purkinje cells decreases in the cerebellum of acrylamide — intoxicated rats, but no cells are lost

The effects of acrylamide intoxication on the numbers of granule and Purkinje cells and the volume of Purkinje cell perikarya have been evaluated with stereological methods. The analysis was carried out in the cerebella of rats that had received a dose of 33.3 mg/kg acrylamide, twice a week, for 7.5 weeks. The total numbers of cerebellar granule and Purkinje cells were estimated using the optical fractionator and the mean volume of the Purkinje cell perikarya was estimated with the vertical rotator technique. The volumes of the molecular layer, the granular cell layer and the white matter were estimated using the Cavalierí principle. The mean weight of the cerebellum of the intoxicated rats was 7% lower than that of the controls rats (2P=0.001). The numbers of the Purkinje cells and granule cells were the same in both groups, but the mean volume of the perikarya of the Purkinje cells in the intoxicated rats was 10.5% less than that of the control group (2P=0.004). The volume of the granular cell layer was reduced by 15% (2P=0.006) but there were no differences in the volumes of the molecular layer and the white matter in the intoxicated and control animals.

The structural effect of systemic NGF treatment on permanently axotomised dorsal root ganglion cells in adult rats

The effect of systemic NGF treatment on loss and shrinkage of dorsal root ganglion cells was studied in adult male rats after permanent axotomy. Nineteen 16 to 18‐wk‐old rats had their right 5th lumbar spinal nerve ligated and cut approximately 7 mm peripheral to the ganglion. Two days before the operation, treatment with subcutaneous injections of human recombinant NGF (1.0–0.5 mg/kg/day) was started in 9 test rats; 10 controls were given saline injections. After 1 mo the levels of substance P (SP) and calcitonin gene related peptide (CGRP) were significantly increased in intact sciatic nerve. The number and mean volume of perikarya were estimated using assumption‐free stereological techniques including vertical sections, the Cavalieri principle, optical disectors, the planar rotator and systematic sampling techniques. Systemic NGF administration had no influence on survival of primary sensory neurons after axotomy. The number of perikarya was 14300 (S.D.=1800) in axotomised ganglia in control rats versus 14700 (S.D.=2100) in axotomised ganglia of NGF treated rats. The reduction of perikarya volume after axotomy was significantly less after NGF treatment (11600 μm3 in the control group versus 8000 μm3 in the NGF treated group). However, the apparent protection of NGF‐treatment on perikaryal volume is explained by a hitherto unrecognised size effect on nonaxotomised dorsal root ganglion cells. The untreated rats had a mean volume of 24700 μm3 (S.D.=2700 μm 3) whereas rats treated with NGF had a volume of 20400 μm3 (S.D.=1700 μm3) on the nonaxotomised side. In conclusion, systemic NGF treatment in adult rats has no effect on dorsal root ganglion cell loss in permanent axotomy whereas perikaryal size of intact nonaxotomised cells is reduced.