Concentración de mercurio (Hg°) en aguas superficiales de la

GAP - 43, a rapidly - transported phosphoprotein of the presynaptic membrane, is a major component of growth cones and is expressesed at high levels during the development of the nervous system especially at the stages when axons elongate (see chapter 1; Benowitz and Routtenberg, 1987; Skene, 1989). Its synthesis and axonal transport persist throughout axogenesis and synaptogenesis, and then decline precipitously with the establishment of stable synaptic relationships (Benowitz et a i,

1981; Skene and Willard, 1981a, b; Kalil and Skene, 1986). GAP - 43 is also present at high levels in mature peripheral neurons of mammals and in lower vertebrate CNS neurons that are regenerating following injury (Skene & Willard, 1981a; Tetzlaff et al.,

1989). The correlation between the ability of axons to regenerate and the increased synthesis and transport of GAP - 43 has given rise to the hypothesis that it may play an important role in axon outgrowth, although the function of GAP - 43 in both normal and growing neurons remains unknown.

The transected optic nerve of adult mammals shows only transient regeneration (see Chapters 3, 4; Hall & Berry, 1989), but injured RGC in adult rats will regenerate axons long distances into a segment of peripheral nerve that has been anastomosed to the cut end of the optic nerve (Berry et a l, 1986a, b; Aguayo et at., 1986). Recent reports in vivo and in vitro have shown that there is an up - regulation of the synthesis and increased axonal transport of GAP - 43 in transected optic nerves of adult rats with or without a PNS graft (Lozano et al., 1987; Doster et al., 1988, 1991; Miotke et al.,

1989), and in the lesioned brain of adult rats with or without a peripheral nerve graft (Oestreicher et al., 1988; Tetzlaff et al., 1990; Campbell et al., 1991). It was suggested that the up - regulation of GAP - 43 depended upon the position of injury; RGC became GAP - 43 immunoreactive only when their axons were injured within 3 mm of the

eyeball (Doster et a l, 1991). On the other hand Jones and Aguayo (1991) showed that GAP - 43 mRNA levels in RGC were elevated in the same degree after both intracranial and intraorbital axotomy. These results are clearly difficult to explain. Furthermore since intracranial optic nerve section leads to many optic nerve fibers dying back into the orbit, many RGC are in fact effectively axotomized in the orbit (see Chapter 4; Richardson et a i, 1982). It would be reasonable therefore to expect such cells to behave like RGC which have been subjected to intraorbital transection. To clarify these matters, I have therefore studied GAP - 43 expression in RGC and in the optic nerve axons after intracranial and intraorbital axotomy using a monoclonal antibody, 9 - IE 12, which reacts with all known forms of the protein (Schreyer & Skene, 1991).

RESU LTS

GAP - 43 IN RETINAL GANGLION CELLS

Intraorbital optic nerve lesion group

At 3 dpo, GAP - 43 - like immunoreactivity was not detectable in the RGC bodies of retinal wholemounts, but by 5 dpo, GAP - 43 immunoreactivity could be visualized in many RGC bodies (Fig. 6.1). The number and intensity of staining of GAP - 43 immunoreactive RGC increased at 7 dpo, appeared to reach its maximum level at 4 weeks (Figs. 6.2 - 5), and then declined sharply by 6 weeks. Some GAP -43 labelled RGC were still detectable at 8 weeks after axotomy (Fig. 6.6). GAP - 43 immunoreactivity was found in RGC with a range of somal sizes. The labelled RGC were distributed evenly in the retinal wholemounts and often showed a highly irregular shape resembling similar axotomized RGC in silver stained preparations of the hamster retina (Cho & So, 1992). The nuclei of many of the large labelled RGC had a very eccentric position (Figs. 6.3 - 4). Axons in the optic fibre layer of the retinas were, however, immunoreactive for GAP - 43 at 3 dpo (Fig. 6.7). The distribution of the axons in the retinas was distinctly radial. The immunoreactivity of the retinal axons within the intraorbital lesion group intensified at 7 dpo, peaked by 4 weeks, and then declined by 6 weeks following operation.

Intracranial optic nerve lesion group

At 3 dpo no RGC bodies were GAP - 43 immunoreactive. Immunoreactive RGC were seen at 5 dpo but they were much less intensely stained than RGC in the intraorbital group after the same interval. Immunoreactive cells were maximum in number at 4 weeks (Fig. 6.8), then diminished by 6 weeks. At 8 weeks, only a few immunostained RGC could be identified (Fig. 6.9). Immunoreactive retinal axons were found at 3 dpo in the intracranial lesion group and remained GAP - 43 positive even 8 weeks following the injury, but they were less intensely stained than in the intraorbitally transected animals. Fig. 6.10 and Table 6.1 shows the number of GAP - 43 labelled RGC in retinal wholemounts following intraorbital and intracranial lesions.

Control groups

To investigate whether the increase in GAP - 43 immunostaining of RGC after intracranial transection of the optic nerve was a consequence of axotomy rather than other factors associated with the surgical procedure, e.g. local trauma to the vasculature, the following experiments were carried out. The optic nerve in some animals was crushed intracranially using forceps to completely axotomize the RGC whilst the integrity of durai sheath was maintained and pial blood vessels were not disrupted. The number of fluorescent labelled RGC in the intracranial optic nerve crush group was not significantly different from the intracranial transection group (133 GAP - 43 immunostained RGC were counted in the intracranial optic nerve crush experiments, compared to 112 GAP - 43 immunostained RGC in the intracranial optic nerve transection). Thus, RGC in both the optic nerve crush and the optic nerve transection experiments become GAP - 43 positive as a consequence of the axotomy.

In other control experiments, in which contralateral retinas were incubated in monoclonal GAP - 43 antibody, the immunostaining of retinal axons was similar to that in introrbital and/or intracranial operated retinas at 3 dpo (Fig. 6.11). However, no immunofluorescent RGC bodies were found at any stage. A quadrant of each retina ipsilateral to the lesion incubated in normal mouse serum instead of anti - GAP - 43

antibody showed neither immunoreactive RGC nor immunostained retinal axons at any stage.

GAP - 43 IN OPTIC NERVE AXONS

Light microscopy

Light microscopic observation of frozen sections showed that GAP - 43 immunoreactivity was localized to the cut end of the optic nerve in the intraorbital lesion experiments. At 3 dpo, immunoreactivity was low and, a few immunostained fibres were present near and at the cut end. Immunoreactivity was higher by 5 dpo (Fig. 6.12) and increased up to 7 dpo (Fig. 6.13). Immunostained axons were present at the cut end of the optic nerve and some extended to the junctional zone between the retinal stump and the distal stump. Immunoreactivity gradually decreased at the cut end from 28 dpo to 56 dpo; however some immuostained axons were still present at the cut end. In addition, some such axons were observed in the distal part of the retinal stump at 42 dpo, and in the more proximal part of the retinal stump by 56 dpo.

In the intracranial lesion experiments, GAP - 43 immunoreactivity was very low and localized to the apex of the degenerative core (described in Chapter 4) at 3 dpo. At 5 dpo, GAP - 43 immunoreactivity was higher and was present at the apex of the core and between degenerative core and the relatively normal surrounding optic nerve tissue (Fig. 6.14), and increased up to 14 dpo (Fig. 6.15). Many immunostained axons extended into the degenerative core from its margins and more from the apex of the core. At 28 dpo GAP - 43 immunoreactivity decreased and was low by 42 dpo. No staining of the degenerative core was apparent at 42 dpo.

Immunoelectron microscopy

Immunoelectron microscopic study of GAP - 43 was performed in animals with intraorbital optic nerve transection at 5 days. GAP - 43 immunoreactivity was revealed by the presence of electron - dense DAB reaction product. Reaction product was located within some medium - sized non - myelinated axonal profiles, but mainly within small

(0.22 - 0.54|im) non - myelinated profiles (Figs. 6.16 - 17) with similar ultrastructural characteristics to those of profiles interpreted as newly regenerated axonal sprouts (see Chapter 3). Reaction product was never found within large non - myelinated and myelinated axons. Immunoreactive sprouts were present in the proximal part of the macrophage zone and the distal part of the abnormal axon zone (see Chapter 3). Some however, were seen within the distal part of the macrophage zone. Immunoreactive profiles were observed within the bundles of axonal sprouts in contact with other sprout - like axons displaying no apparent immunoreactivity. However, some small clusters or individual immunoreactive sprouts were scattered in the extracellular spaces (Fig. 6.18). They were round in cross - sectional profile, contained microtubules, mitochondria, elements of smooth ER, vesicles and sometimes floccular cy to skeletal components. Immunoreactive sprouts were seen in contact with astrocyte processes; some, however, were in contact with astrocyte cell bodies, with macrophages and with fibroblasts. Occasionally, they were observed in contact with oligodendrocytes (Fig. 6.19).

The ultrastructural localization of reaction product within the axonal sprouts was associated with the plasmalemma, with microtubules, and with the outer surfaces of vesicles and mitochondria (Figs. 6.16 - 17). However reaction product in some axonal sprouts appeared to be associated with the axon cytoplasm.

No GAP - 43 immunoreactivity was detected in control material in which the primary antibody was replaced by noiTnal mouse serum. The optic fibres on the unoperated side displayed no immunoreactivity by light or electron microscopy.