Evaluación económica del sistema de abastecimiento de agua por bombeo con

Freeze killed grafts did not promote the same regenerative response as those containing living Schwann cells (see Chapter 4). This implies that the living cells in the graft produce growth promoting factors which can enhance axonal regeneration or affect neuronal size. Denervated Schwann cells are known to upregulate their production of NGF, which is one such factor. In this chapter (i) the synthesis of this neurotrophin by these grafts, once implanted into the adult rat corpus striatum, and (ii) the upreguladon of receptors for this molecule by host neurons are investigated.

ra)Summarv of methods used See Chapter 2 for details:

Autologous tibial nerve grafts were implanted into the right striatum of adult Sprague Dawley rats. After 7 days fresh samples of graft and brain tissue from these and control unoperated rats were analysed for NGF content using a 2-site ELISA immunoassay.

The presence of p 7 5 ^ ° ^ low affinity NGF receptor protein in similar grafted striata was assessed at 14 days po using immunohistochemistry.

(b) Results

Enzvme-linked immunoassay of NGF levels in the graft and surrounding brain tissue Tissue samples were taken from animals 7 days after surgery.

Animal numbers indicate whether different tissues were taken from the same or different animals.

Table 5.1 shows the estimated level of NGF determined for each of the structures listed using a 2-site ELISA technique and measured in pg/ul. At 7 days po, sections of autologous tibial nerve graft implanted into the neostriatum of adult rats were found to contain much higher levels of NGF than the surrounding striatum. The highest concentration of NGF was found in the graft tissue with little difference between the levels of NGF found in either the portion of the graft within the brain or that which remained outside.

The expression of low affinitv NGF receptor p 7 5 ^ ° ^ protein bv adult striatal neurons in response to graft implantation

Striatal neurons in the unoperated rat or contralateral to the grafts did not express immuohistochemically detectable levels of p75^*^^. Up-regulation of p 7 5 ^ ° ^ was not seen in any striatal perikarya in response to the implantation of a tibial nerve graft 14 days earher (Fig. 5.1). The presence of immunoreactivity in cholinergic medial septal neurons acted as a positive control for this technique (Fig. 5.2b), confirming that the lack of immunopositive neurons in the striatum was not the result of a faulty visualisation procedure. The Schwann cells of the graft were also strongly immunopositive for this protein and this enabled the demonstration of the linear orientation of the Schwann cell columns within the graft (Fig. 5.1 and 5.2 a and c). There was no evidence of Schwann cell migration into the brain. p75^^°^ positive neuropil was seen 'lining' the graft/brain interface but no immunopositive neuronal cell bodies were identified in the striatal tissue around the graft. Some p 7 5 ^ ° ^ immunopositive neurons were commonly seen in the more caudal sections (Fig. 5.3a). These neurons were always found in regions of the ventral pallidum and medial forebrain bundle and were present whether or not the graft had distui'bed the area during implantation. In aU such cases neurons with similar perikaryal size and morphology were found in the corresponding location in the contralateral hemisphere (Figs. 5.3b and c).

T IS S U E S P E C IM E N ANIMAL N o . W E IG H T ( g ) N G F LEVEL (pg/mg) M EA N N G F LEVEL ( pg/mg) EX T ER N A L CW106 0.0076 178 2 3 9 G R A FT CW107 0.0072 225 CW108 0.0058 266 CW109 0.0062 232 CW210 0.004 235 CW211 0.0029 296 INT E R N A L CW106 0.0026 308 2 9 0 G R A FT CW107 0.003 313 CW108 0.0047 282 CW109 0.0036 341 CW210 0.0032 231 CW211 0.0026 269 IPSIL A T E R A L CW106 0.058 23 2 4 STR IA T U M CW107 0.039 32 CW108 0.0278 24 CW109 0.0274 35 CW210 0.0318 18 CW211 0.0332 14 C O N T R A L A T E R A L CW106 0.062 9 12 ST R IA T U M CW107 0.034 10 CW109 0.0302 18 CW210 0.031 9 CW211 0.032 12 ISOLATED C W lll 0.0082 129 1 3 3 SEGMENT OF CW112 0.0110.0 134 PERIPHERAL _{CW113 081 136} NERVE LEFT I N S IT U TIBIAL NERVE C W lll 0.0164 143 1 4 9 CUT AND CW112 0.0104 142 LIGATED; CW113 0.0137 145

DISTAL SEGM ENT CW114 0.0139 167

Table 5.1 The level of NGF contained in graft and striatal tissue samples 7 davs po as determined bv enzvme linked immunoassay

Figure 5.1

rCW85^

Immunohistochemistry for low affinity NGF receptor. G = graft, S = striatum, MS = medial septum,

Arrows = p75^°^'^ immunopositive Schwann cells.

At 14 days po no p 7 5 ^ ° ^ immunopositive neuronal perikarya were seen in the striatum surrounding the graft although high back ground staining was present. However, Schwann cells within the graft itself were strongly immunopositive for p7^LNGFR leaking their linear arrangement within the graft apparent. The Schwann cells at the proximal tip of this graft show very good contact with the striatal tissue but there was no evidence of Schwann cell migration out of the graft into the brain. Scale bar = 100p.m.

Figure 5.2

Immunohistochemistry for low affinity NGF receptor. G = graft, S = striatum,

Arrows = p 7 5 ^ ^ ^ immunopositive Schwann cells,

Arrow heads = p 7 5 ^ ^ ^ immunopositive neuronal perikarya, MS = medial septum.

5.2a (CW851 Another example of immunonegative striatal neurons around strongly immunopositive PN graft at 14 days po. Scale bar = lOOjim.

5.2b rCW861 Strongly immunopositive neuronal perikarya in the medial septum of the same animal acting as a positive control for the effectiveness of the antibody. Scale bar = 10|im._

5.2c (CW85^ Detail of boxed area in Fig. 2a, discrete immunopositive Schwann cell columns can be seen within the graft, particularly at the graft/brain interface. Again, although background staining is high around the graft, no immunopositive neuronal perikarya are visible. Scale bar = 10|im.

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5 .2 c

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MS

Figure 5.3

Immunohistochemistry for low affinity NGF receptor. G = graft, S = striatum,

1= ipsilateral brain, C = contralateral brain,

Arrows = p 7 5 ^ ° ^ immunopositive Schwann cells,

Arrow heads = p 7 5 ^ ^ ^ immunopositive neuronal perikarya.

5.3a (CW99) An example of a strongly p75^*^^ immunopositive neuron, with a large cell body and branching primary processes, in the caudal medial striatum a small distance from the graft. Due to its caudal medial location within the striatum this cell was thought to be one of the more rostral p75^^^^ immunopositive neurons of the ventral pallidum/basal forebrain areas. Scale bar = 100p.m.

5.3b and c ('CW99) p75^'^'^ immunopositive neurons of the ventral pallidum/ basal forebrain areas which could be found in corresponding positions close to the caudal medial striatum with similar intensity of staining both ipsilateral (I) and contralateral (CS) to the graft. Scale bar = 100pm.

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5.3b

5.3c

CHAPTER 6

H ypertrophy of cholinergic striatal neurons ipsilateral to a