Longitud y Pendiente del Área de Muestreo

This study demonstrates that hsp27, hsp60 and hsp72 mRNA levels increase in the first 24 hours of permanent MCAO in the core region o f ischaemia. The constitutive mRNA levels o f hsp27 and hsp70 were almost undetectable. Hsp27 and hsp72 mRNAs were significantly increased against to and contralateral controls as early as 2 hours post-occlusion. Hsp60 levels on both the Northern and slot blots demonstrated a significant increase at 8 hours post-occlusion. All of these mRNA levels further increased at 24 hours. Increases in the protein levels o f these hsps followed at subsequent time points, hsp27 and hsp60 were first detectable at 24 hours, and the hsp72 increase being first detectable at 8 hours. No constitutive levels o f hsp27 and 72 proteins were detected. Hsp72 is the inducible member of the hsp70 family, but hsp27 is present at low levels in neurons. The low level o f detection o f hsp27 protein in the ipsilateral cortex at 24 hours, despite the high increase in mRNA induction may be representative o f the binding o f the monoclonal antibody which was raised against the Chinese hamster ovary hsp27 species, and therefore may not bind with high affinity to the rat species.

Hsp 5 6 mRNA levels did not increase but the protein levels did exhibit a mild increase at 24 hours. The hsp90 mRNA levels were not as clear-cut. Although the levels were variable on the graphic representation of the slot blot data, hsp90 mRNA levels appeared to be increased in conq)arison to the P-tubulin mRNA levels at 8 and 24 hours on the Northern blot against to and contralateral controls. The slot blot data demonstrated significantly greater levels in the ipsilateral core against the to controls at 8 and 24 hours, but was only significantly increased against the contralateral controls at 24 hours. No increases in the concentration o f hsp90 mRNA can definitively be concluded m this study. The hsp90 protein levels were not notably increased against the to or contralateral controls but any increase in protein as a result o f the possible increase o f mRNA at 24 hours would not, on the evidence o f the other hsp profiles, be detected until afl;er this time point.

This is the first study that examines the levels of hsp5 6 mRNA and hsp5 6, hsp60 and hsp90 protein levels in any model of cerebral ischaemia. This is also the first study of overall mRNA and protein expression o f hsp27, hsp56, hsp60, hsp70, and hsp90 mRNA and protein levels exclusively in the core region o f focal cerebral ischaemia. Higashi et aL, examined the mRNA levels o f hsp27, hsp47, hsp70 and hsp90 during permanent MCAO, but the extraction for Northern blot analysis was taken fi'om whole brain shces, rather than exclusively the core region (Higashi et aL, 1994). In agreement with our data, Higashi et al. demonstrated an increase in hsp27 mRNA levels up to 24 hours, whereas they also demonstrated that hsp 70 mRNA levels declined afl;er 16 hours occlusion. They also stated that hsp90 mRNA levels were not raised throughout the timecourse, although the data was not presented. Transient global ischaemia (10 minutes) increases the mRNA levels o f the a-isoform o f hsp90 in the gerbil hippocampus with the highest levels at 8 hours post-occlusion, continuing before the levels diminished at 2 days (Kawagoe et aL, 1993a). The cDNA probe and antibody used in this study detect both isoforms o f hsp90. The data iu this study suggests that hsp 90 mRNA and protein levels are not significantly increased in the core region in the first 24 hours o f focal cerebral ischaemia. Hsp60 mRNA levels have only previously been investigated in the gerbil hippocampus afl;er transient global ischaemia (Abe et aL, 1993a). A 3.5 minute occlusion stimulated an increase in hsp60 mRNA after 3 hours post-occlusion and was sustained until 24 hours in the CAl neurons, and a mild increase was noted in the CA3 neurons at 3 hours. Hsp60 mRNA levels, therefore, are increased as a result o f ischaemia, and this study has demonstrated that levels also increase during a severe ischaemic insult to cortical tissue and that this increase is followed by a subsequent increase in protein levels.

The areas o f gerbil hippocan^us that undergo delayed neuronal death, such as the CA l subfield, show an inhibition of overall protein synthesis, throughout the recovery period from 5 minutes o f global ischaemia (Thilmann et aL, 1986). The other areas of the forebrain demonstrate a transient inhibition of protein synthesis, with ^H-tyrosine incorporation reaching normal levels between 30 minutes and 12 hours post-ischaemia, and this includes the region suppHed by the middle cerebral artery. Protein synthesis during stress is believed to be impaired in part due to defects in spHcing, and the

increases in hsp70 mRNA demonstrated in the CAl region during ischaemia may be attributable to the lack o f introns in the gene. Permanent focal cerebral ischaemia is a more severe insult than global, evident by the widespread irreversible brain injury. The data generated by this study has demonstrated that in the most severely affected region of cortex the levels o f certain hsp mRNA and protein levels are still increasing 24 hours after MCAO. Unfortunately, no material from ischaemic tissue was available later than 24 hours occlusion, as it would have been interesting to study the levels at further time points.

This study is limited, in that no locahsation of expression has been defined. This could be achieved by hnmunostaining for hsp protein expression and in situ hybridisation for mRNA expression on ischaemic brain shces. Previous work has gone some way in illustrating the sub-locahsation o f expression o f the hsps during global and focal cerebral ischaemia. The expression of hsp27 protein has been largely locahsed to the astrocytes and microgha of the ipsilateral hemisphere from 4 hours reperfusion after 1 hour o f transient MCAO (Kato et aL, 1995). There was also a corresponding difRise immunostaining for hsp27 in the gha in non-MCA territory in the ipsilateral hemisphere, including the midbrain, thalamus and hippocampus, and some in the contralateral hemisphere. One study has demonstrated that hsp is transferred from gha to neuronal axons (Tytell et aL, 1986). This might suggest that during ischaemia, the gha rnay offer some protection to neurons in repair and restoration after ischaemia through neuron-gha interactions. Increases in hsp70 protein hnmunostaining after permanent MCAO has been locahsed to the endothehal cehs in the infarct and to neurons in the ischaemic penumbra outside the infarct, whereas increased hsp70 mRNA has been locahsed to the penumbra both inside and outside the infarct (Kinouchi et aL, 1993b; Kato et aL, 1995).

The purposes, if any, o f these increased expression profiles have not been defined, and are therefore speculative. Conditions that increase hsp levels have been demonstrated in cerebeUar granule and cortical cehs to protect against the cytopathic effects of increased extracehular glutamate, such as occurs during ischaemia (Abe et aL, 1993a; Rordorf g/ aL, 1991). The increased presence o f hsp70 hnmunostaining as a result of

global and focal ischaemia may only be a by-product o f ischaemia and a marker of neurons or gha that have survived by an alternative mechanism Hsp 70 may, however, be directly responsible for the survival o f these cells. Dorsal root ganghon cehs overexpressing hsp 70 are resistant to ceU death in vitro as a result o f simulated ischaemia (increased lactic acid in the presence of sodium dithionite at pH6.5), although the protection is not as profound as that which it confers against ‘lethal’ heat shock (Amin et aL, 1996).

Although heat shock protects neurons in vitro against ap opto sis, neuron-derived ceh lines and trigeminal neuronal cultures overexpressing hsp70 or hsp90 have been demonstrated not to be protected against the apoptotic stimuh o f serum- withdrawal/retinoic acid and nerve growth factor withdrawal, respectively (Mailhos et aL, 1993; Mailhos et aL, 1994; Wyatt et aL, 1996). Therefore, any protection conferred in vivo may only be against the necrotic mechanisms induced by ischaemia.

Although many fimctions have been attributed to the various members of the hsp70 family (targeting non-native proteins for proteosomal digestion, uncoating clathrin baskets, maintaining non-native proteins in an unfolded state for bilayer translocation) the function(s) o f the inducible hsp72 (hsp70i) are unclear. All the members of the hsp 70 family bind to denatured proteins, therefore the inducible member may bind to proteins in the cytosol and nucleus that have misfolded or denatured as a result of

stress and prevent their aggregation, enable correct refolding during recovery, and target denatured proteins for digestion or bilayer translocation to enable correct folding in cellular organelles.

Overexpression of hsp27 protects against ‘lethal’ heat shock, but no in vitro or in vivo evidence has been presented to suggest that it may protect against ischaemia. Its role as an actin binding protein may serve to stabihse the cytoskeleton during stress, enabling recovery. Overexpression also blocks the apoptotic mechanisms stimulated by staurosporine and the Fas/APO-1 receptor in murine fibrosarcoma cells (Mehlen et aL, 1996). Ap opto sis is one of the mechanisms of cell death as a result of ischaemia, and therefore overexpression in the gha may confer a similar protection to these cehs. The

increased levels o f hsp27 in the glia in the ischaemic core and penumbra, and surrounding cortex, may promote survival of the gha during the stress and recovery. Astrocytes maintain the ionic and neurotransmitter concentrations o f the extracellular fluid and therefore survival of these gha may be important for restoration o f these concentrations to prevent cytopathic effect, for example as a result o f glutamate toxicity. I f hsp27 is transported flrom the gha to neuronal axons, then it may serve to stabihse the structural integrity of these neurons and enable recovery. Hsp27 also has a role as a molecular chaperone. It has been demonstrated thus far to enable the folding of citrate synthase and a-glucosidase, and may therefore chaperone other proteins, possibly preventing protein aggregation and misfolding and enabling correct protein folding during recovery ifrom ischaemia.

Hsp60, the mitochondrial chaperonin and eukaryotic homologue o f the prokaryotic GroEL molecule has not been demonstrated to confer tolerance to heat shock in cehs, despite its levels being raised during ceh stress. Its function has thus far been defined as providing an environment within its core to enable the correct folding o f mitochondrial proteins. The increase noted in this study of mRNA and protein levels as a result of permanent MCAO and in the subregions o f the hippocampus after transient global ischaemia may, like hsp72 and hsp27, serve a purpose in preventing ceh death as a result of ischaemia. Recovery of correct mitochondrial protein folding may be facihtated by raised levels of hsp 60.

Dorsal root ganghon neurons overexpressing hsp90 in vitro are resistant to ceh death under TethaT heat shock conditions, but any resistance to simulated ischaemia was not as profound as that when hsp70 is overexpressed (Amin et a/., 1996). Like hsp70, hsp90 overexpression does not confer any protection against ap opto sis (Mailhos et a/., 1994; Wyatt et aL, 1996). Any increase in expression o f hsp90 may therefore confer some protection against necrotic ceh death during ischaemia, possibly by chaperoning misfolded proteins such as actin, tubulin or signal transduction molecules, preventing their aggregation, and enabling correct folding on recovery. Hsp90 and hsp5 6 may act by binding to the untransformed steroid receptor conq)lex, maintaining its correct

In summary therefore, these data show definite increases in the levels o f hsp27, hsp60 and hsp70 mRNA and protein levels within the first 24 hours o f focal cerebral ischaemia in the rat. The other hsps tested (hsp90 and hsp56 mRNA and protein) did not appear to be significantly raised.

In considering these results as a whole, it is clear that for greater understanding, further investigations into the role o f the hsps in cell death as a result of ischaemia are required before the hsps, individually or in concert, can be named as the mediators of protection that is observed in the 'ischaemic tolerance’ phenomenon. It may therefore be usefiil to deliver the genes encoding these heat shock proteins to the brain, and assess whether overexpressing hsps in vivo affords any protection to neurons and gha during MCAO.

Chapter 4

Construction and Characterisation of