Estudio de Site Survey en Planta de Producción

3000-1 Control PI P2 o u im O o E § S 1000- j - 2000- Apo 100 150 200 250 300 0 50 Time (mins)

Fig. 3.10a - Responses of 3-NP and METH-treated rats to apomorphine (Apo, 0.5 mg/kg s.c.)

Effects of apomorphine on rats treated with different

combinations of 3-NP and METH

Control P5 P6 P7 , * * * o Ç 1000- Apo * * * 100 150 200 250 300 0 50 Time (mins)

Fig. 3.10b - Responses of 3-NP and METH treated rats to apomorphine (Apo, 0.5 mg/kg, S .C .). (***p<0.001 versus control)

________________________ Chapter Three Neurochemical basis o f the 3-NPM model 3.2.1.5 Indications from the preliminary studies

3-NP is a common neurological toxin that prevents myelination of nerve fibres (Gould and Gustine, 1987) and induces cell death by inhibiting mitochondrial respiratory processes (Hamilton and Gould, 1987). When small doses are administered in isolated instances, the body appears to be quite capable o f coping, as seen by the apparently normal levels of DA and 5-HT in all four brain regions of the 3-NP controls. In paradigms PI, P2 and P3, 3-NP was administered at a dose of 2x15 mg/kg, at 6 h intervals for one, two and three days respectively. As would be expected, when the titre of the toxin in the body built up (following repeated administration), the detoxifying mechanisms became overwhelmed and neurodegeneration escalated. Whereas striatal DA depletion was at 55 % in PI, it rose to 72 % in P3. The same trend is observed with 5-HT. It was therefore of benefit, in subsequent experiments, to increase the dose and fi'equency of administering the toxin, as well as to reduce the number of days between the administration of 3-NP and METH.

While it is generally accepted that an underlying energy deficiency exists in the elderly and that such a deficiency could accentuate any neurotoxic insult, there is no consensus on whether the impaired energy state is the consequence of a previous traumatic experience. This is because there is no evidence as yet to limit the cause of idiopathic PD to an exogenous toxin. If the culprit trigger factor for the disorder is some genetic deficiency or age-induced impairment in energy metabolism, then that would fit in quite well with the endogenous toxin view. This is because a reduction in ATP level has been shown to render the DA neurones susceptible to physiological levels of endogenous excitatory amino acids (Zeevalk et al, 1995, Sonsalla et al., 1998). On the other hand, the

Exogenous Toxin theory will be more applicable if it can be demonstrated that administration of the said toxin would give rise to a cascade of events that will culminate in the inhibition of

mitochondrial Complex I, and the ultimate death of DA cell bodies in the SN pars compacta of the nigrostriatal tract. This appears to be the case with the exogenous, synthetic toxin, MPTP. METH is not a known inhibitor of mitochondrial respiration.

Going by the Endogenous Toxin theory, we hypothesized that administering an appropriate dose of 3-NP at a suitable time prior to the administration of an ineffective dose of METH would achieve a synergism that would ultimately yield DA cell death in the SN and terminal loss in the ST.

________________________ Chapter Three_____Neurochemical basis o f the S-NPM model

the METH dose of 4 x 8 mg/kg, every 2 h the next day (P5 on the index of modes, table 2.1). This yielded only 26 % DA depletion in the ST, with no apparent change in the other brain regions assayed. However, this dosing schedule was sufficient to accomplish 100 % 5-HT depletion in the ST and 93.5 % depletion in the SN. Conversely, when METH was administered prior to a lower dose of 3-NP (10 mg/kg, given daily for four days), P6, a 72 % depletion in striatal DA and 46 % depletion in NAcc DA levels was observed (Fig.3.5 and 3.2 respectively). Bearing in mind that P6 involved repeating the dosing schedule, the level of striatal DA loss is comparable to the 69 % striatal DA depletion in P4, which was a repetition of the P5 dose schedule after two weeks. The main difference between administering METH before or after 3-NP, is the greater damage to 5-HT neurones when they are rendered vulnerable to METH by pre-treatment with 3-NP.

In the behavioural experiments (section 3.2.1.4), the 3-NP control rats showed high spontaneous activity with a greater resistance to habituation. This may correspond to the hyperactive state suggested by Borlongan et al., 1997. However, these rats showed no DA receptor supersensitivity as revealed by the low scores in response to the apomorphine challenge. The spontaneous

locomotor activity o f rats from P4 and P6 was markedly low, but nowhere near the rigidity

observed in reserpine-treated rats. All the rats showed no apparent limitation in locomotor activity, and no gross changes in behavior were observed across the paradigms. The only apparent change was seen in the extent of response to apomorphine. The rats from P5 and P7 showed significant increase in locomotion, suggesting DA receptor supersensitivity arising from DA loss.

In a bid to reduce the length of time involved in producing a functional model, while minimizing fatalities, the dose of 3-NP in P4 was reduced from 40 to 20 mg/kg, and administered for four consecutive days prior to METH (i.e. P7). This yielded a 67 % reduction in striatal DA (Fig.3.5) and a significant depletion of 5-HT only in the SN (Fig.3.8). In an attempt to further reduce

nigrostriatal tissue DA levels, the inherent benefits of P6 and P7 were harnessed. This gave rise to a dosing schedule where 3-NP (20 mg/kg, s.c.) was administered once daily for four consecutive days, followed by METH (4 x 5 mg/kg, q2h, i.p.) on day five, and the 3-NP daily dose for the succeeding four days. The dose of METH was reduced to 4 x 5 mg/kg to reduce fatality. Behaviour experiments were conducted five to seven days later (but typically on day five), and the rats were sacrificed on the seventh day after the last injection of 3-NP. This is the “3-NPM” model.

________________________Chapter Three Neurochemical basis of the 3-NPM model