Proceso de fabricación de puertas tipo acordeón

There are a number of reasons why the vitamin E deficient animals might not have developed diarrhoea despite the increase in basal and secretogogue induced electrogenic secretion. Firstly, the findings in the jejunum do not necessarily reflect what is happening in other regions of the small and large bowels, as has been made clear in other studies of electrogenic secretion in the rat^^®'^^°, and secondly, the enormous capacity of the rat caecum renders this species highly resistant to diarrhoea. It was noticeable that the E- animals were more likely to have a distended caecum than E+. Performing a caecectomy on the animals at an earlier age might have allowed the development of clinical diarrhoea.

11.5.1.1 Use of vitamin E deficiency as a model of increased free radical fluxes within the intestine

Vitamin E deficiency was used as a means to increase free radical fluxes because this seemed appropriate to a real life situation which may occur in malnourished infants. Vitamin E, as discussed in previous chapters, is an important structural

element of the cell membrane in addition to being the most important lipid soluble antioxidant. Hence, this animal model is best regarded as a model of both vitamin E depletion and increased free radical fluxes. Ionising radiation has been used by other groups to provide an oxidising stimulus within the intestines^^\ but this is somewhat unphysiological and it was considered an inappropriate stimulus for the purposes of the present studies. The small intestine is, infact, more susceptible to radiolysis induced oxidative injury than to other types of oxidative stress^^^'^^^'^^^, and this observation discouraged me further from its use. Other options which were available as a means of increasing oxidative stress included ischaemia / reperfusion injury^®^ and the addition of exogenous oxidising stimuli®^®. Reservations about the use of ischaemia / reperfusion injury and the technical problems encountered with this stimulus in the rat have been discussed in chapter 10 and will not be discussed further.

The addition of an oxidising stress in vitro in the Ussing chamber has been used by a number of other groups. Don Powell's group demonstrated an increase in

electrogenic Cl secretion and a reduction in electroneutral Na and 01 absorption in rat colon following the generation of hydrogen peroxide on the serosal surface of the tissue using xanthine / xanthine oxidase^^®. These alterations in ion transport were largely ( - 70%) due to the release of prostaglandins from within the lamina propria. No measurements were made of peroxidative injury to the intestinal mucosa. Interestingly, Desjeux's group in Paris have described an electrogenic secretory response to serosal HgOg in rat small intestine which became greater when rats were fed a protein deficient diet®^®. These studies did not provide any evidence that these responses were due to oxidative damage within the small intestinal mucosa. The electrogenic responses to HgOg were small in the fed group of rats (-7 pA cm'^). I was unable to demonstrate any change in Isc following the application of Im M serosal HgOg to jejuna from the rats used in my studies.

11.5.1.2 Problems with the animal model

There are a number of potential problems with the animal model as used with this particular cohort of animals. These are discussed in turn below.

11.5.1.2.1 Nutritional status and dietary intake of the animals

The E- group ate significantly less food than the E+ group, suffering in effect chronic undernutrition in comparison with their age matched controls. This may have influenced both the electrophysiological and the biochemical measurements (vide infra). For the purposes of this preliminary study I chose not to pair feed the animals. If vitamin E deficiency induced a state of anorexia (as does vitamin A deficiency) then it seemed appropriate, at least in the first instance, to allow "nature to take its course". Had the animals been pair fed then the question would have arisen whether they should have been fed on the basis of food per rat, or food per kg of body weight. Indeed, it would have been interesting to have seen what would have happened to the weights of the deficient animals had they been pair fed (on a food per rat basis) to the intake of the sufficient group.

Acute undernutrition (33% of normal intake for 9 days) has no effect on basal Isc in stripped duodenum, jejunum or ileum, but is associated with an increased electrogenic secretory response in jejunum to the Ca^" mobilising agonists bethanecol®^\ 5 hydroxytryptamine, and prostaglandin Eg®^®, and to E coli STa (which activates secretion via the guanylate cyclase / cyclic GMP pathway) but

not to dibutyryl cAMP and forskolin (which cause secretion via cyclic AMP dependent mechanisms). These patterns of jejunal hypersecretion are different from those found in this cohort of vitamin E deficient rats. The effects of prolonged malnutrition on these parameters have not been reported. Before one can be sure that the changes in intestinal short circuit current documented above are due to vitamin E deficiency per se rather than due to starvation / undernutrition appropriate studies with pair fed animals need to be performed. Studies in animals with similar dietary intakes are described in a later chapter.

11.5.1.2.2 Vitamin A status

Purkins et al^^° used a diet which was similar to that used in these studies [the "control" diet contained 100 mg a tocopherol / kg feed], and noted a significant fall in plasma vitamin A concentrations in weanling rats fed the diet for six months. The reasons for the fall in plasma vitamin A were not sought. In a previous cohort of vitamin E deficient rats in our laboratory, plasma concentrations of vitamin A fell by a median of 58% (95% Cl 5-78%, n=5 P<0.05) in the E- group by 12 months of age when compared with age matched vitamin E sufficient rats®. Nzegwu et

have studied the effects of chronic vitamin A deficiency on rat small and large intestinal secretion and absorption. They found that vitamin A deficiency did not alter basal Isc in the jejunum, and resulted in a fall in Isc in the distal ileum. Electrogenic glucose transfer was not affected by vitamin A deficiency although bethanecol produced a significantly larger increment in Isc in the jejunum of vitamin A deficient animals than in controls. It seems unlikely therefore from this data that the changes in electrogenic absorption and secretion in this vitamin E deficient cohort of animals could be attributed to vitamin A deficiency per se. Small intestinal

concentrations of vitamin A were also similar in both E deficient and sufficient animals. Intestinal concentrations of vitamin A were not reported by Nzegwu et a P .

11.5.1.2.3 Immune regulation of secretion

Chronic vitamin E deficiency is associated with a modulation of the immune system, including a reduction in the number of circulating T helper cells in ra ts ^ and in other animals^^^. Products of lipid peroxidation, for example 4 - hydroxynonenal, have a potent chemoattractant effect on granulocytes^^'’. The immune system has effects on epithelial ion transport^^®’^^® and perturbations of the immune system may therefore lead to modulation of intestinal secretion. These effects have not been specifically studied in the current rat model. Histological sections of the small intestine from vitamin E deficient animals did not demonstrate a hypercellularity of the lamina propria as might be expected if there were an augmented inflammatory response in this group of animals.

11.5.1.2.4 Enteric neuropathy

Chronic vitamin E deficiency in both man®®^ and this animal model is associated with a characteristic neuropathy of the peripheral, central, and autonomic nervous systems®®®’®®®’. The enteric nervous system has a regulatory role in the control of intestinal ion transport and the contribution of an enteric neuropathy to the observed changes in electrogenic secretion remains undefined. A number of observations may be made which are pertinent to this matter. Firstly, basal

intestinal short circuit current in the rat small intestine (in contrast to rat colon) is not normally influenced by neural blockade with serosal tetrodotoxin®^® ®^’ . The lack of change in Isc after neural blockade with TTX in the E deficient animals in the present studies provides evidence against a direct role of the enteric nervous system in the generation of these higher basal short circuit currents. Matters may, however, be more complex in undernourished animals. Rats starved for three days exhibit increased stores of tyramine releasable noradrenaline in the enteric nervous system®^®. The effects of catecholamines in the neural regulation of small intestinal secretion are modulated in vitamin E deficiency and this is discussed further in chapter 12. The histochemical studies performed by Charles Hoyle failed to provide any structural evidence of a neuropathy in the vitamin E deficient jejuna. A number of attempts were made to study the electrogenic secretory response to

’ Charles Hoyle, Department of Anatomy, University Coiiege, London Unpubiished data

electrical field stimulation in this group of animals, but consistent results were difficult to obtain in jejunum. The enteric nervous system has a role in the electrogenic secretory response to STa^^^ but this seems to relate more to the duration of the response than to its initial magnitude. [Both these parameters will be of importance because it is the area under the curve which relates to the size of the secretory response.] The peak response to STa was increased in the E deficient animals, but the duration of the responses were similar in the two groups of animals. From the evidence presented above it seems unlikely that an alteration in the level of neural tone in the E deficient jejuna was directly responsible for the increase in basal Isc.

11.5.1.2.5 Tissue capacitance

It is not possible to entirely rule out an alteration in the capacitance of deficient jejuna when compared with the sufficient animals. This has the potential of leading to an underestimation of short circuit current in the "bulkier" E+ tissues. It would be surprising however if the difference in capacitance were sufficiently large to explain the differences between the two groups given the relatively small differences in tissue resistance and in tissue thickness.

11.5.2 Lipid peroxidation

Oxidative injury to the small intestinal mucosa was measured both as TBARS and as MDA. As discussed in chapter 3, these assays are complimentary in the information they provide. The findings of increased concentration of TBARS and of free and total malondialdehyde in the E- jejuna are consistent with a greater degree of oxidative damage in E- jejuna than in E+. The concentration of TBARS in the intestine are quite modest when compared with concentrations found in other tissues such as liver^^^ This may be due to both the increased resistance of the small bowel to oxidative damage and also the relatively low amounts of PUPA In the intestine when compared with other tissues^^^.

It is well established that lipid peroxidation has effects on the protein conformation of the brush border membrane^^®, on adrenoreceptors^^®, and on the physical properties of the biological membranes®^\ Changes in the lipid environment of transporter proteins is also known to have an effect on the functioning of these proteins®^®.