IV. RESULTADOS Y DISCUSIÓN
4.5. APLICACIÓN DEL MODELO SEAMOD A LA CUENCA DEL RÍO HUANCANÉ
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CONTRALATERAL SIDE GRAFTED SIDE
H istogram 7.1 Comparison of striatal NADPH-diaphorase positive perikarval areas ipsiiateral and contralateral to a living tibial nerve graft over time.
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2 WF.F.K SURVIVAL 5 1 3 1 1 1 1 ig O O C OO (N ^ ^ ^ . - ^ (N (N (N ro rn § o o o o o ■stU .o o o ^■sf <n A o o o - o- g o g g o o o OOCNVOO-stOOfN _ OO -st N E U R O N A L AREA (^im2) G R A F T E D SIDE C O N T R A L A T E R A L S ID EHistogram 7.2 Comparison of striatal NADPH-diaphorase positive perikarval area ipsiiateral and contralateral to a freeze-killed tibial nerve graft at 2 weeks po.
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CHAPTER 8
Expression of GAP-43 mRNA and the synthesis of GAP-43 protein bv adult striatal and nigral neurons ipsiiateral and contralateral to an
autologous peripheral nerve graft
Intrinsic differences between populations of CNS neurons may affect their ability to mount a regenerative response after injury either independently of, or in addition to, their ability to respond to any exogenous trophic support offered by peripheral nerve grafts. This chapter compares the expression of GAP-43, a growth associated protein, in neurons of the striatum and substantia nigra pars compacta of the adult rat, ipsiiateral and contralateral to peripheral nerve grafts, two regions which display very different regenerative responses.
(a') Summarv of methods used See Chapter 2 for details:
Autologous tibial nerve grafts were implanted into the right striatum of adult Sprague Dawley rats. Sections of unfixed striatal and nigral tissue were processed for in situ hybridization using an oligonucleotide probe for mRNA for the growth associated protein GAP-43. The sites of mRNA synthesis were established 5 days, 10 days and 1 month after graft implantation. Similar observations were made using an immunohistochemical technique to visualize GAP-43 protein in sections from perfused animals at the same survival times. The position and appearance of any GAP-43 immunopositive sprouts and axons in the striatum and graft was recorded.
(b) Results
In situ hybridization for GAP-43 mRNA
In situ hybridization identified a population of GAP-43 mRNA containing neurons in both the corpus striatum and substantia nigra of the normal, unoperated adult rat and the control contralateral striatum. The striatal GAP-43 mRNA positive neurons were few in number, weakly labelled and scattered evenly throughout the body of the striatum; all had conspicuously large perikarya (Fig. 8.1a and b). Thus as already been shown by McKinney and Kent, (1994), the striatal GAP-43 mRNA positive ceUs correspond to the large striatal aspiny intemeurons. The heavily labelled GAP-43 mRNA positive nigral neurons, which were found only in a diagonal swathe of cells corresponding closely with the position of the substantia nigra pars compacta (Fig. 8.2 a and b), match the perikarya of the dopaminergic nigro-striatal projection neurons described by Kruger et al. (1993).
The implantation of a tibial nerve graft into the corpus striatum of the adult rat seemed to have no modulatory effect on the expression of GAP-43 mRNA in either of the two regions at any of the survival times examined. Figure 8.1 a and b shows the appearance of these cells ipsiiateral and contralateral to a tibial nerve graft at 10 days po. GAP-43 mRNA expression appeared to differ httle either between survival times or between the grafted and control sides of the brain. However, it is probable that the heavy labelling characteristic of normal adult neurons of the SNpr may have masked upregulation of GAP-43 mRNA in response to implantation of the graft.
Immunohistochemistrv for GAP-43 protein
No perikaryal GAP-43 protein could be detected in either the corpus striatum or substantia nigra when the above experiments were repeated using immunohistochemistry (Fig. 8.1c and d). GAP-43 positive sprouts were seen around the graft/brain interface at 10 days po, and within the graft itself (Fig. 8.3 c - f). Figures. 8.3 a and b show the course of an immunopositive axon, which can be traced for 160|im within the striatum, oriented towards the graft at 10 days po. This axon terminated in a strongly immunopositive fan-like structure (small arrow) which was interpreted as a growth cone due its morphology and the high level of GAP-43 protein visuahsed within it.
The only examples of up-regulation of either the protein or mRNA for GAP-43 were seen in a few isolated cortical cells located within 200|im of the injury tract caused by implantation of the graft. Fig. 8.4 a and b show the position and appearance of two distinctly immunopositive cortical cells found at 10 days po while figure 8.4c and d illustrates cortical up regulation of GAP-43 mRNA visualised by in situ hybridization at 5 days po.
Figure 8.1
No upregulation in GAP-43 mRNA or protein synthesis was seen in response to graft implantation by 10 days po.
G = graft, S = striatum, CS = contralateral striatum,
Arrow heads = GAP-43 mRNA containing or immunopositive neuronal perikarya.
8.1a (CW18D No GAP-43 immunopositive neuronal perikarya were found in the striatum ipsiiateral to the graft at 10 days po. Scale bar = 100p.m.
8.1b (CW18D Contralateral striatal sections showed no immunoreactivity for GAP-43 protein. Scale bar = 100pm.
8.1c CCW235) A small population of large striatal neurons are known to contain GAP- 43 mRNA (McKinney and Kent, 1994) and were visible using in situ hybridisation, however no other population of striatal neurons were seen to up-regulate their synthesis of this molecule in response to graft implantation. Scale bar = 100pm.
8.Id CCW235) In the contralateral striatum, GAP-43 mRNA was only seen in a small population of large striatal neurons similar to those seen in Fig. 8.1c. Scale bar =
8.1a
8.1b
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Figure 8.2
SNpc = substantia nigra pars compacta, SNpr = substantia nigra pars reticulata,
Arrow heads = GAP-43 mRNA containing neuronal perikarya.
8.2a rCW204) At 5 days po the same pattern of GAP-43 mRNA containing neuronal perikarya were found in the SNpc on either side of the brain of these animals regardless of graft placement in the ipsüateral or contralateral striatum. Scale bar = 100|im.
8.2b (CW2361 No difference was seen in nigral GAP-43 mRNA expression in similar grafted animals at 10 days po. Scale bar = 100p.m.