Luisa Perasso

Therapeutic Use of Creatine in Brain or Heart Ischemia: Available Data and Future Perspectives

Creatine (Cr) is essential in safeguarding ATP levels and in moving ATP from its production site (mitochondria) to the cytoplasmic regions where it is used. Moreover, it has effects unrelated to energy metabolism, such as free radical scavenging, antiapoptotic action, and protection against excitotoxicity. Recent research has studied Cr‐derived compounds (Cr benzyl ester and phos‐pho–Cr–magnesium complex) that reproduce the neuroprotective effects of Cr while better crossing the neuronal plasma membrane and, hopefully, the blood–brain barrier (BBB). Intracellular levels of Cr can be increased by incubation with Cr or some of its derivatives, and this increase is protective against anoxic or ischemic damage. A large amount of experimental evidence shows that pretreatment with Cr is capable of reducing the damage induced by ischemia or anoxia in both heart and brain, and that such treatment may also be useful even after stroke or myocardial infarction (MI) has already occurred. Cr has been safely administered to patients affected by several neurological diseases, yet it has never been tested in human brain ischemia, the condition where its rationale is strongest. Phosphocreatine (PCr) has been administered after human MI, where it proved to be safe and probably helpful. Cr should be tested in the prophylactic protection against human brain ischemia and either Cr or PCr should be further tested in MI. Moreover, Cr‐ or PCr‐derived drugs should be developed in order to overcome these molecules’ limitations in crossing the BBB and the cell plasma membrane.

In vivo neuroprotection by a creatine-derived compound: phosphocreatine-Mg-complex acetate.

Phosphocreatine-Mg-complex acetate (PCr-Mg-CPLX) is a creatine-derived compound that in previous in vitro research was able to increase neuronal creatine independently of the creatine transporter, thus providing hope to cure the hereditary syndrome of creatine transporter deficiency. In previous research we showed that it reproduces in vitro the known neuroprotective effect of creatine against anoxic damage. In the present paper we investigated if PCr-Mg-CPLX reproduces this neuroprotective effect in vivo, too. We used a mouse model of transient middle cerebral artery occlusion. Mice received PCr-Mg-CPLX or a mixture of the two separate compounds phosphocreatine (PCr) and MgSO(4), or vehicle. The injections were done 60 min and 30 min before ischemia. Forty-eight hours after ischemia neurological damage was evaluated with Clarks behavioural tests, then the infarct volume was measured. PCr-Mg-CPLX reduced the infarct volume by 48%, an effect that was not duplicated by the separate administration of PCr and MgSO(4) and the neurological damage was decreased in a statistically significant way. We conclude that PCr-Mg-CPLX affords in vivo neuroprotection when administered before ischemia. These results are comparable to previous research on creatine administration in experimental stroke. PCr-Mg-CPLX maintains creatine-like neuroprotective effects in vivo as well as in vitro. Our study suggests that PCr-Mg-CPLX might have a therapeutic role in the treatment of hereditary creatine transporter deficiency and of conditions where there is a high risk of impending stroke or cerebral ischemic damage, like high-risk transient ischemic attacks, open heart surgery, and carotid surgery.

Systemic Administration of Mesenchymal Stem Cells Increases Neuron Survival after Global Cerebral Ischemia In Vivo (2VO)

Although many studies have shown that administration of stem cells after focal cerebral ischemia improves brain damage, very little data are available concerning the damage induced by global cerebral ischemia. The latter causes neuronal death in selectively vulnerable areas, including the hippocampal CA1 region. We tested the hypothesis that intravenous infusion of bone marrowderived stromal cells (mesenchimal stem cells, MSC) reduce brain damage after transient global ischemia. In adult male Sprague-Dawley rats transient global ischemia was induced using bilateral common carotid artery occlusion for 20 min in addition to controlled hypotension. Five days after, the animals were anaesthetized with urethane and the brain was fixed, sectioned and stained with hematoxylin-eosin to investigate histological damage. MSC did not fully protect against ischemic damage, as the number of viable neurons in this group was lower than in normal (sham-operated) rats. However, in MSC-treated rats the number of viable CA1 pyramidal neurons was significally higher than in rats that had been subjected to ischemia but not treated with MSC. We conclude that intravenous administration of MSC after transient global ischemia reduces hippocampal damage.

A novel hypothesis about mechanisms affecting conduction velocity of central myelinated fibers.

The hypothesis that gap junctions are implicated in facilitating axonal conduction has not yet been experimentally demonstrated at the electrophysiological level. We found that block of gap junctions with oleammide slows down axonal conduction velocity in the hippocampal Schaffer collaterals, a central myelinated pathway. Moreover, we explored the possibility that support by the oligodendrocyte to the axon involves energy metabolism, a hypothesis that has been recently proposed by some of us. In agreement with this hypothesis, we found that the effect of oleammide was reversed by pretreatment with creatine, a compound that is known to increase the energy charge of the tissue. Moreover, conduction velocity was also slowed down by anoxia, a treatment that obviously decreases the energy charge of the tissue, and by ouabain, a compound that blocks plasma membrane Na/K-ATPase, the main user of ATP in the brain. We hypothesize that block of gap junctions slows down conduction velocity in central myelinated pathways because oligodendrocytes synthesize ATP and transfer it to the axon through gap junctions.

Action observation: mirroring across our spontaneous movement tempo

During action observation (AO), the activity of the “mirror system” is influenced by the viewer’s expertise in the observed action. A question that remains open is whether the temporal aspects of the subjective motor repertoire can influence the “mirror system” activation.

Effects of exogenous creatine on population spike amplitude and on postanoxic hyperexcitability in brain slices

In in vitro rat hippocampal slices, a short period of transient anoxia caused a lasting increase in the amplitude of the compound action potential (population spike, PS) that was evoked in CA1 by stimulation of the Schaffer collaterals. No such increase was seen over a comparable period of time in slices that were not subjected to anoxia. The appearance of such an increase was dependent on the duration of anoxia. Anoxia of 1 min duration did not cause any increase, anoxia lasting 2 min caused a nonsignificant increase, while 3 min of anoxia caused a lasting and statistically significant increase in PS amplitude. Addition of creatine, a compound that is known to afford protection against severe neuronal damage from longer periods of anoxia, prevented PS potentiation at a concentration of 10 mM, but not at a concentration of 1 mM. In addition, while 1 mM creatine by itself did not show any effect on PS amplitude of control slices, 10 mM creatine decreased PS amplitude also in such control slices, that had not been exposed to anoxia. These data demonstrate that this postanoxic hyperexcitability is caused by mechanisms that are little sensitive to the protection that in other contexts is provided by creatine. We suggest that understanding the mechanisms of postanoxic hyperexcitability may help understand the pathophysiology of the epileptic seizures that sometimes occur at the time of an ischemic stroke.

IVGTT-based simple assessment of glucose tolerance in the Zucker fatty rat: Validation against minimal models

For the assessment of glucose tolerance from IVGTT data in Zucker rat, minimal model methodology is reliable but time- and money-consuming. This study aimed to validate for the first time in Zucker rat, simple surrogate indexes of insulin sensitivity and secretion against the glucose-minimal-model insulin sensitivity index (SI) and against first- (Φ1) and second-phase (Φ2) β-cell responsiveness indexes provided by C-peptide minimal model. Validation of the surrogate insulin sensitivity index (ISI) and of two sets of coupled insulin-based indexes for insulin secretion, differing from the cut-off point between phases (FPIR3-SPIR3, t = 3 min and FPIR5-SPIR5, t = 5 min), was carried out in a population of ten Zucker fatty rats (ZFR) and ten Zucker lean rats (ZLR). Considering the whole rat population (ZLR+ZFR), ISI showed a significant strong correlation with SI (Spearman’s correlation coefficient, r = 0.88; P<0.001). Both FPIR3 and FPIR5 showed a significant (P<0.001) strong correlation with Φ1 (r = 0.76 and r = 0.75, respectively). Both SPIR3 and SPIR5 showed a significant (P<0.001) strong correlation with Φ2 (r = 0.85 and r = 0.83, respectively). ISI is able to detect (P<0.001) the well-recognized reduction in insulin sensitivity in ZFRs, compared to ZLRs. The insulin-based indexes of insulin secretion are able to detect in ZFRs (P<0.001) the compensatory increase of first- and second-phase secretion, associated to the insulin-resistant state. The ability of the surrogate indexes in describing glucose tolerance in the ZFRs was confirmed by the Disposition Index analysis. The model-based validation performed in the present study supports the utilization of low-cost, insulin-based indexes for the assessment of glucose tolerance in Zucker rat, reliable animal model of human metabolic syndrome.

Calpain-1 resident in lipid raft/caveolin-1 membrane microdomains plays a protective role in endothelial cells

We are here reporting that calpain-1 is a constitutive component of a distinct lipid raft/caveolin-1 microdomain isolated from bEnd5 cells in association with endothelial nitric oxide synthase (eNOS) and heat shock protein 90 (HSP90). Perturbations in intracellular calcium concentration by Ca2+-ionophore A23187 or prolonged cell exposure to high glucose induce a significant decrease in the level of eNOS accompanied by a recruitment of additional HSP90 molecules at this site. In these conditions the cells are more resistant to cell death by Ca2+ overload. The decrease of eNOS has been due not only to its Ca2+-mediated release from the caveolin-1 aggregates but also to its digestion by calpain-1. The specific involvement of calpain-1 in digestion of eNOS is supported by the preventive effect of a synthetic calpain inhibitor (CI-2) and by the absence of calpain-2 and calpastatin in the caveolin-1 microdomain. These results suggest that the protein adjustments observed in lipid raft/caveolin-1 microdomains could be visualized as a process required to protect the cells against NO overproduction and aberrant calpain activation. Alterations in eNOS, calpain-1 and HSP90 levels have been observed in aorta of Zucker Diabetic Rats (ZDR). The loss of HSP90 occurring in these animals indicates an aberrant activation of calpain and thereby the transition from a physiological to a pathological cell condition.

Simple Assessment of Insulin Sensitivity in the Zucker Rat

The model-based assessment of insulin sensitivity in Zucker rat from Intravenous Glucose Tolerance Test (IVGTT) data is a common procedure. The minimal model methodology provides a very reliable assessment but requires specific competence for running the model. The aim of this study was presenting calculated SI (CSI), as a surrogate index for the simple assessment of insulin sensitivity in the Zucker Rat from IVGTT data. To this aim 25 Zucker Lean Rats (ZLR) and 25 Zucker Fatty Rats (ZFR) were considered. Reference insulin sensitivity (SI) was estimated in each rat through the minimal model methodology. CSI is defined as the ratio between the rate of glucose disappearance (KG) and the mean supra-basal area under the insulin curve during the test (ΔAUCINS), corrected by the proportionality term, α. Regression analysis between SI and KG/ΔAUCINS was performed to identify the α coefficient. Results showed as the computed value of CSI presented a high correlation (r = 0.89, R-square = 0.80 and p < 0.0001, slope ≈1) with SI. Mean value of CSI over the whole population was not significantly different from correspondent SI value (p = 0.17). CSI is able to detect the wellknown reduction of insulin sensitivity in the ZFR group (1.0±0.1 vs. 5.0±0.7 min-1/μU·ml-1, p < 0.001), in accordance with the results provided by SI. In conclusion, the present study proposes CSI, as a suitable empiric index for a simple and reliable assessment of insulin sensitivity in Zucker rat and able to provide the same quantitative information of modelbased SI.