Effect of ionising radiation on the axon reaction of mouse anterior horn motor neurons. A histological and immunocytochemical study using a monoclonal antibody to neurofilament protein.

The changes taking place in irradiated central nervous tissue prior to the onset of delayed radionecrosis are poorly understood, but functional abnormalities occurring during the latent interval after irradiation are likely to be of importance. In order to investigate functional disturbances in neurones during this period, unilateral sciatic nerve crush was performed in mice following sub-lethal X-irradiation of the lumbar spinal cord. Alterations in the axon reaction of anterior horn cells were studied using a monoclonal antibody to neurofilament protein. With irradiation immediately prior to crush, the normal, well-defined increase in perikaryal neurofilament protein was significantly diminished, although there was no concurrent radiation necrosis and no alterations were seen in contralateral neurones with intact distal axon processes. The effect was more marked in neurones irradiated one month prior to nerve crush, and in the non-irradiated nerve crush region regeneration was delayed, with diminished neurofilament protein in the regenerating axons. These observations indicate that ionising radiation can progressively impair the ability of neurones to synthesise neurofilament protein during distal axon regeneration. This may result from inadequate repair of radiation induced DNA strand-breaks, but may also follow more generalised damage to protein transcription enzymes and RNA metabolism.

Langerhans cell number and morphology in mouse footpad epidermis after X irradiation.

Effects of 250-kV X rays on epidermal Langerhans cells were studied in CBA/CaH mice. One group received 20 Gy to the feet, another 8 Gy to the whole body, and a third both the 8 Gy whole-body and a 12 Gy local dose to the feet. Mice from each group and controls were sacrificed at intervals from 1 to 64 days later. ATPase-positive cells in sheets of footpad epidermis were counted by light microscopy. The density of Langerhans cells in controls was 1515 +/- 36/mm2 (mean +/- SE; n = 34). By 3 days after irradiation they became rounded and less dendritic and numbers gradually reached a nadir by 10 days, at 18% of controls after 20 Gy and 57% of controls after 8 Gy. Some of the remainder exhibited bizarre morphology and ultrastructural abnormalities. After local irradiation of the feet Langerhans cell numbers recovered rapidly between 14 and 16 days, although their distribution was uneven until 30 days after irradiation. Repopulation was delayed after an 8 Gy whole-body dose by at least 3 weeks. These results demonstrate that high local doses of X rays substantially but transiently deplete the epidermal Langerhans cell population and support the hypothesis that functional hemopoietic tissue is required for extensive Langerhans cell replenishment.

Incidence of radiation-induced skin tumours in mice and variations with dose rate.

Mice were exposed to weakly penetrating beta-particles from an external source, using 12 different surface doses ranging from 5.4 to 260 Gy and given at four different dose rates from 200 to 1.7 cGy/min. As in previous investigations, both epidermal and dermal tumours occurred with the latter predominating. The lowest surface dose to produce a statistically significant increase in skin tumours was 21.7 Gy, no effect being detected with doses of 5.4-16.3 Gy. The dose-response curves rose steeply when obvious increases occurred. Consideration of these findings and the fact that radiation-induced skin tumours can have an exceptionally long latent period leads to the suggestion that there is some relatively radioresistant factor which normally restrains potential radiation-induced cancer cells in the skin from becoming tumours until the skin is subjected to high local doses. Tumour-induction was unaffected by reducing the highest dose rate by a factor of 10 and the dose-response curves were almost identical. Further reductions of dose rate, encompassing a further factor of 10, in general resulted in fewer tumours.

The axon reaction in motor and sensory neurones of mice studied by a monoclonal antibody marker of neurofilament protein.

Following unilateral sciatic nerve crush in mice, changes in the neurofilament content of neuronal perikarya were studied, using a monoclonal antibody to neurofilament protein (RT97). In the spinal cord, anterior horn motor neurones, normally unstained, showed a positive staining reaction with immunoperoxidase on the operated side. This reaction was short lived and maximal on the 11th post-operative day. In spinal ganglia, the proportion of positively staining sensory neurones showed an earlier but otherwise similar increase. In both cases, the response was well defined and contrasted with the changes on Nissl staining, which were markedly different in the two populations of neurones. In the nerve crush region, although regenerating axons were visible with silver staining only 5 days post-operatively, neurofilament protein was not demonstrated in these axons until several days later, after the peak perikaryal increase. These results suggest that an increase in perikaryal neurofilament protein is a consistent and quantifiable event following distal axon trauma, possibly indicating either synthesis of protein subunits or repolymerization of neurofilaments prior to their transport distally down the regenerating axons. The findings may be useful in identifying neurones with distal axon lesions in experimental and other neuropathological material.

Quantitative light and electron microscopic studies on the ventral roots of the wobbler mutant mouse.

In wobbler mutant mice and normal litter-mates the numbers of axons have been counted in cervical and lumbar ventral roots and the dimensions of nerve fibres measured in the fifth cervical, C5, and third lumbar, L3, roots. In wobbler cervical ventral roots C5, C7 and C8 and lumbar roots L4 and L5 the number of axons was reduced. Abnormal features were seen in all mutant cervical ventral roots but not in the lumbar roots. The histogram of fibre size was shifted to the left in the wobbler C5 roots where axon loss was considerable, particularly amongst the large diameter axons, and in L3 roots where, although there was no over-all loss of axons, there were fewer ventral root fibres of large diameter. Abnormal features were also observed in a few dorsal root ganglion cells.

A comparative scanning electron-microscopical study of endoneurial collagen around normal mouse nerve fibres, nerve fibres following crush injury and nerve fibres of the dystonic mouse mutant (dt/dt).

The endoneurial collagen sheath around teased nerve fibres following crush injury was studied by scanning electron microscopy and compared with uninjured sciatic nerve fibres and with fibres from the dystonic mutant mouse. Following crush injury the endoneurial collagen became more abundant than seen in untreated nerve fibres and formed large, seperate and longitudinally oriented bundles. However, by four weeks post injury the sheath regained a normal external appearance. Mutant nerve fibres were also associated with more than the usual amount of collagen, but the sheaths were more disorganised, with a marked disorientation and irregular aggregation of collagen, and these abnormalities were not confined to obviously degenerating or demyelinated regions of the fibres. The dystonic abnormalities of the endoneurial sheath may be important in the mechanism of the neuropathy.

A rapid method for visualising nerve terminal innervation in human muscle using an acetylcholinesterase method combined with silver staining.

A modified acetylcholinesterase-silver technique has been used to study the nerve terminal innervation in human muscle removed at biopsy and in post-mortem material. The results of "Functional Terminal Innervation Ratio's" are given, and the advantages of using this method are discussed.

Motor innervation of the gastrocnemius muscle of wobbler and dystrophic mice.

Innervation of the gastrocnemius muscle of mice from the wobbler, dystrophic and C57/BL colonies has been studied. It was found that phenotypically normal mice from each of the colonies did not differ in their innervation properties, hence suggesting no heterozygote penetrance. However, the end-plate complexity increased with age of normal mice. Functional terminal innervation ratio for both the wobbler and dystrophic gastrocnemius muscle was raised above that of the normal and many dystrophic end-plates also appeared abnormal. A study of mice from the wobbler colony manifesting a late onset hind-limb muscle degeneration (Wr/HLD) has been included and the results suggest a relatively benign form of spinal muscular atrophy.

The relationship of Schwann cell migration in vitro to injury, using normal, Wobbler, and dystrophic mice.

A quantitative in vitro study of the cellular outgrowth from mouse sciatic nerve explants has been carried out using both normal untreated nerves and nerves taken at various times after the in vivo application of trauma. The results obtained have been compared with the results for outgrowth from sciatic nerve of two neurological mutants: 'the Wobbler mouse' and the 'ReJ 129 Dystrophic mouse'. It has been shown that the in vitro response to peripheral injury does reflect the activity known to occur in vivo. Outgrowth from explants of mutant mouse nerves, although differing from normal has been found to be less profuse than that occurring after mechanical nerve injury.