Model of Global Permanent Ischemia in Conditions of Normothermia and Hypothermia

We propose a model of global permanent ischemia on rats, obtained through decapitation of the animals...

Other studies have also shown that the same threshold of tolerance of a neuronal population might be changed allowing recovery of the ability of protein synthesis or interrupting the dissolution process of intracellular organelles [6,7].
Alongside with the progresses in analyzing the pathogenesis behind the ischemic insult, neuroprotection mechanisms have become matter of interest. In particular studies on all those methods, pharmacological, physical or chemical that can reduce the biochemical examinations. At the end of each period of thermic treatment, the brains were immediately immersed in liquid nitrogen and processed to obtain a deproteinized tissular extract, to be used for the definition of the biochemical parameters selected through high-performance liquid chromatography (HPLC) technique.
The brains were taken from the liquid nitrogen and immediately homogenized for 60 seconds in the presence of perchloric acid (HCIO 4 ) 1.2 M cold, so to obtain the complete deproteinization of the specimen. The Homogenate of each specimen obtained in the abovementioned way has been centrifuged for 15 minutes at 13000rpm (at 4°C); the supernatant was separated and stored at 4°C, while the pellet obtained was newly homogenized with HCIO 4 1.2M cold.
After a further centrifugation (13000rom/10 minutes, 4°C), the new supernatant was added to the previous of each specimen and the whole was neutralized through the addition of K 2 CO 3 5M. Following a new centrifugation (13000 rpm/10 minutes, 4°C), the pellet of sunken salt was discarded and an equal volume of chloroform (1:1; v:v) was added to the neutralized tissular extract, in order to obtain the removal of all the non-water-soluble components present in the specimen. After having whirled the specimen for 60 seconds, a centrifugation of the specimen at 10000 rpm for 10 minutes at 4°C was carried out; the superior aqueous phase thus obtained was drawn (composed then exclusively by the aqueous supernatant free from any lipoid leftover) which was stored at -80°C until the time of the HPLC examination, before which the specimen was accurately filtered with a 0.45μM MILLIPORE-HV filter.

Biochemical Parameters of the Research
On the neutralized acid tissular extracts of rat brain, the

HPLC Chromatographic Method of Examination
HPLC consists in a small modification of the previous ionic coupling methods perfected in our labs, during which the tetrabutylammonium is used as a coupling agent. A rate of the aqueous phase (100μl) will be examined through high performance liquid chromatography (HPLC) whose apparatus consist of a double pump system ThermoQuest Constametric 3500 (ThermoFinnigan Italia, Rodano, Milano, Italia) connected to an in series-diode spectrophotometric developer Spectra System UV6000LP (ThermoFinnigan Italia) which was set between the wave lengths of 190 and 330 nm. All this system is, in turn, connected to a PC for the acquisition and analysis of data, which uses a software system supplied by the same company that produces the HPLC system.
For the analysis of the specimen an analytic chromatographic column Kromasil C-18, 250 mm x 4.6 mm was used, with 5μM pores (Eka Chemicals, AB, Bohus, Sweden) provided with the specific precolumn. The column was balanced with a mobile phase (A tampon) containing Hydroxide tetrabutylammonium 10 nM, KH 2 PO 4 10nM, and methanol at 0.25%, with 7.00 pH. A gradient "with leaps" was carried out through a second tampon (B) containing hydroxide tetrabutylammonium 2.8 nM, KH 2 PO 4 100mM, and methanol at 30%, with 5.50 pH; the gradient used is the following: 15 minutes at 100% of A; 5 minutes at 90% of A; 5 minutes at 70% of A; 15 minutes at 63% of A; 15 minutes at 55%; 3 minutes at 50% of A; 32 minutes at 48% of A; 10 minutes at 28% of A; 15 minutes at 0% of A. The flux of the chromatographic runs was 1.2 ml/min and the temperature were maintained constant at 23°C, through a specific thermostatic system. The separation of the compounds in the experiment, which are identified through the confrontation of runs of ultra-pure standards both of the respective retention times and of the absorbing spectrums, has permitted the calculus of the concentrations carried out at the specific wave lengths of each substance.

Statistical Analysis of Data
The results of the different experiments were analyzed with specific statistical tests as the two-tailed Student test-t for noncoupled specimen.

Results
The results emerged from the experiments refer to the rat It is important to notice that, following the extraction of the specimen in percloric acid, there is the total transformation of the compounds reduced in the correspondent oxidative form. Hence, in the particular case of the nicotinic coenzymes it is indicated with the acronym NAD (or NADP) the total concentration of NAD+NADH (or NADP + NADPH). It is interesting to note that the reduction of NAD levels ( Figure 4) could be correlated to the parallel increase of the concentrations of ADP ribose ( Figure 5) which, in turn, is the expression of the activity of the poly (ADP-ribose) polymerase (PARP) system. The correlations of the two occurrences will be examined in the discussion phase.          for the transformation; in such a way the production of free radicals is consequently favored. It is important to note that, as it is inferred by the reported reaction, the formation of free radicals is a process that requires the availability of oxygen. In the present experimental model of global ischemia such availability is a function of a certain quantity of oxygen leftover at the tissue level and of the quota that the same tissue can acquire from the atmosphere.
This allows, albeit more controlled, for the production of those toxic substances. In our opinion, this is a particularly important concept because it reaffirms what is many times stated in other parts of this dissertation: the production of free radicals is a process typical of the reperfusion period that is nevertheless in part influenced by mechanisms that begin to activate themselves before the restoration of the hematic flux. Also in vivo it is actually possible that one quota of leftover oxygen is available at the cellular level; furthermore, the data correlates perfectly with the models of temporary focal ischemia, in which the oxygen can spread through diffusion towards the infarct areas from the adjacent zones still perfused. In such conditions an increase of the formation of free radicals happens, even before the occlusion at the base of the situation of ischemia is removed [22,23]. Among the different ways of formation of the oxygen derivate, the one that plays an important role in the phase of ischemia in the strict sense is the reaction mediated by the XO, while the others activate only at a later stage when the hematic flux is reestablished [24,25].
As evidence of what has been stated, in the present model we observe a progressive raise of the concentrations of uric acid which represents the final product of the reaction previously described.
Considering then the trend of the MDA levels (  [24,25] previously described, it has emerged that the maximum peak of formation of the two isoforms of the NOS shows up at the distance of 24 hours from the ischemic event. It is true that from that research it has also emerged that the intraischemic hypothermia (33ºC) was able to reduce the activity and cellular expression of the iNOS also at a distance of only 2 hours from the beginning of the ischemic event, but is also true that the model used in that case was one of a temporary focal ischemia and that the sample taken after 2  [26]. Such a process seems to be the result of the previously described mitochondrial alterations that occur in the first hours after the ischemic insult and that see involved, as a main responsible, the intracellular calcium [27]. Nevertheless, from the analysis of the results of this research emerges also another data that could be correlated to the reduction of the levels of the nicotinic coenzymes: the progressive increase of the quota of ADPribose. The ribosylation of the ADP following the ischemic event is a process mediated almost exclusively by the action of the PARP system [28,29].
These cellular enzymes are activated through only one known system, represented by the single filament breaking of the DNA chain [30]. The alterations on account of the cellular nucleic acids are the result of the free radical actions: the peroxynitrite first and foremost, but also the hydroxyl radical and the superoxide are the compounds mainly involved [31]. Nevertheless, the registered raise of the ADP-ribose levels would seem to support it. If so should be, the progressive reduction of the NAD concentrations could be interpreted also in light of a direct action by the PARP system on the nicotinic coenzyme, since these enzymes are able to provoke a cleavage of the NAD, becoming the main responsible of its depletion in conditions of focal ischemia [29]. Furthermore, this model would demonstrate that an activation of the PARP system, maybe even partial, can happen even while in were not recorded in the present experimental model, the reduced concentrations of ADP-ribose documentable at 31 and also at 34ºC, cannot be interpreted on the base of a minor activation of the enzymatic system subsequent to a lower damaging action at the nuclear level from the oxygen derivate; it is therefore more likely that the hypothermia is capable to directly antagonize the action of the enzymes, once that these are normally activated.
It is fair to underline that at 34ºC, together with a reduction of the ADP-ribose concentrations mainly comparable to the one that happens at 31ºC, a significant difference of the NAD levels compared to those recorded at 37ºC is not evident; the data should not come as a surprise, since it is to be remembered that in conditions of ischemia other mechanism exist that can provoke the reduction of the NAD levels, different from the cleavage action exercised by the PARP system. It is sufficient to think that the synthesis of the nicotinic coenzyme is an ATP dependent process and that it is carried out partly inside the mitochondria that begin to present marked alterations of their structure and function already after an hour of the beginning of the ischemia [32,33].
Moreover, as it has been demonstrated by other researches [7,34], the NADH molecule, just like the NADPH one, are another preferred

Conclusion
Hypothermia is indeed one of the most important neuroprotective measures and through this animal model it was possible to demonstrate how even after 2 hours from an ischemic event a certain value of energetic metabolites still available remains.
In addiction it seems that this protective effect might be due to the activity of the PARP system. Finally, metabolism interruption seems to be not an instantaneous process but rather something occurring in a longer period of at least 2 hours.