We induced transient focal brain ischemia by unilateral infusion of Et-1 near the middle cerebral artery. This model offers two advantages: (i) a gradual reperfusion that closely mimics the rate of reperfusion in humans; and (ii) the induction of ischemia without the counfounding effects of anaesthesia. Anaesthesia affects blood pressure, blood gases and body temperature, which might influence stroke severity and outcome, including the mortality rate. This might contribute to explain the lower mortality rate found in both young and middle-aged rats (3.7% and 8.3%, respectively), as compared to values reported in the monofilament or embolic blood clot models of MCA occlusion (6% or 12% for 3-month-old rats, and about 22% and 35% for 12-month and 22-24-month rats) [18, 31].
Sensorimotor deficits in ischemic rats were reminiscent of those observed in humans after stroke . One of the most remarkable similarities is the clenching of the affected forepaw fingers and snuggling of this forepaw towards the torso, which we often observed in the PHR test. The behavioral tests used for assessment of asymmetry, i.e., the postural hang reflex, limb placing, limb use asymmetry (cylinder) and adhesive tape removal tests are commonly used for the evaluation of neurological deficits after transient ischemia in rodents [23–27, 33–36]. The early PHR test, which we performed as an inclusion/exclusion criterion , demonstrated that the Et-1 model could be applied with success to middle-aged rats (excluded only 3 out of 27 ischemic rats) and, therefore, is valuable as a translation model for the evaluation of neuroprotective strategies in human. Most of the studies show that in advanced aged rodents (22-24-months) the outcome of stroke, in terms of infarct size, mortality, behavior, and somatosensory functions, is severely impaired with respect to young rats (References in ). However, there are also reports of greater histological damage in young than in old rats, and reduced behavioral deficit in aged rats after embolic stroke (intra-carotid injection of microspheres) . Here we did not find differences in infarct volumes in the two age groups. To our knowledge there are only few studies using middle-aged rats with transient occlusion of MCA [15–18]. Middle-aged (12-month) rats have been shown to acquire stronger neurological motor impairment in beam walking test than young rats (3-month) after photothrombosis of the hindlimb sensorimotor cortex . In another study, robust, reliable and long-term behavioral deficits were found in 16-month-old rats subjected to MCA occlusion, although no comparisons were made with young rats . No differences in ischemic volume and in the percentage of cerebral blood flow reduction in the parietal cortex were found between young and middle aged rats subjected to filament occlusion of MCA . An increase in infarct volume and attenuation of ischemia-induced striatal neurogenesis were reported in 12-month-old rats with filament MCAO as compared to 3-month-old rats . After Et-1 infusion in anaesthetized rats, age was found to have a distinct influence on functional recovery, with 10-days old rats showing a greater potential for plasticity than 6-months old rats . The present study is the only one hitherto that focuses on the middle age period of life (12 month) in freely moving rats subjected to Et-1-induced occlusion of the MCA. We observed greater and longer-lasting asymmetry in ipsilateral-contralateral sensory-motor performance in middle-aged ischemic rats as compared to young ischemic rats for the entire period of observation (14 days after stroke induction). Tests assessing limb use (support on a cylinder and adhesive tape removal) were more sensitive in revealing differences in left-right asymmetry related to aging than the postural hang reflex and limb placing tests. The more pronounced behavioral asymmetry in middle-aged rats with respect to young rats was due to a greater age-dependent impairment of somatosensory functions in the contralateral side of the body in response to ischemia. Having in mind the deleterious impact of age per se on the whole organism, a reasonable hypothesis is that old rats (24-27-month old) subjected to ischemia would have worse outcomes than middle-aged rats but this has not been investigated with the Et-1 model as yet. Using the filament occlusion model, comparisons between outputs in old and middle-aged rats are reported in only few studies. In particular, a greater deficit score was reported in 18-month than in 12-month rats, and degenerative morbidity and mortality was shown in 24-month rats . A greater infarct volume in 18-month than in 9-month rats , and no difference in infarct volume between 12-month and 18-month rats were also reported .
We implemented EEG measurements simultaneously with behavioral observations in an attempt to explain behavioral asymmetry. Only a few studies have examined bioelectrical activity after unilateral infusion of Et-1 in long-term recordings [12, 14, 20, 37]. Here we examined EEG asymmetry by means of the pair-wise Brain Symmetry Index, which measures the asymmetry in power spectral density along homologous cortical channel pairs of both hemispheres [29, 38]. These studies showed that pdBSI may be of value in assisting visual interpretation of EEG during carotid endarterectomies, in correlating clinical severity in patients with ischemic cerebrovascular disease (transitory ischemic attacks, stroke), and in monitoring patients with anterior circulation syndrome of presumed ischemic origin. Importantly, the degree of EEG asymmetry of the sensorimotor cortex between lesioned and non-lesioned corticospinal systems at long times after the onset of stroke in humans is inversely related to recovery of motor function . This strengthens the importance of mechanisms of homeostatic plasticity during recovery from focal, unilateral stroke. Analysis of the interhemispheric EEG asymmetry in models of focal ischemia in rodents has not been carried out, as yet. Recently, the BSI index approach was used in swines to show the utility of quantitative EEG for diagnosis and prognosis of cerebral arterial gas embolism . In their short-term EEG investigation after embolization (240 min), the authors have shown that temporal BSI correlated with intracranial pressure, brain lactate and brain oxygen tension. In the present study, we showed that pdBSI in young rats increased shortly after Et-1 (at 15 and 60 min) compared with recordings before Et-1, while in aged rats pdBSI after ischemia was greater than pdBSI before ischemia or pdBSI in young rats during the whole experimental period (up to 14 days post-Et-1). These finding suggest that middle-aged rats were more vulnerable to Et-1-induced ischemia than young rats.
The regional analysis of spectral density EEG profiles showed in both aged groups a suppression of the EEG power (except for the slowest frequencies) in the ipsilateral somatosensory areas during the acute period of ischemia, which paralleled the behavioral deficits. A relationship between reduction of fast-band EEG power in the affected hemisphere and impairment of hand functionality was reported in humans . The increase in the EEG power of the slow delta wave components in ipsilateral somatosensory cortex at H4 and again at D3 coincided with the periods of reperfusion and established brain damage during the chronic phase, respectively . The alterations in the oscillating cortical slow activity in the ipsilateral somatosensory cortex during and after the reperfusion phase of Et-1-induced ischemia might be due to reoxygenation of the ischemic tissue, which is known to provide chemical substrates for intracellular mechanisms leading to neuronal death and neurological deficits . Oxidative stress-related generation of toxic compounds, such as free radicals, reactive oxygen or nitrogen species during the reperfusion phase  may be detrimental to neurons, thereby causing EEG abnormalities. A close association between EEG/MEG abnormalities and some markers of the oxidative stress or free radical scavengers (iron, peroxides and transferrin) have been found in patients with Alzheimer disease  and stroke . It will be a challenge to trace a path from the molecular events associated with oxidative stress during ischemia to the excitability of the cerebral cortex at the EEG level after stroke in experimental animals and humans.
We also found changes in the EEG power distribution in the contralateral somatosensory cortical areas, which were opposite to those seen in the ipsilateral areas in the ischemic middle-aged rats: while in the ipsilateral cortex the EEG was suppressed during the acute phase of ischemia, in the contralateral cortex the EEG power was increased. This is in line with data obtained in rodents  and humans  after stroke. Numerous studies have examined the role of the contralateral cortex in functional recovery after unilateral stroke. Contralateral synchronized neuronal activity occurs as a remote effect of the primary ischemic lesion (the diaschisis phenomenon) one to three days after stroke in experimental animals . In humans, ischemia-induced neuronal reorganization, which extends to the contralateral areas, is supposed to be associated with interhemispheric asymmetry of inhibition . Neuronal sprouting, formation of new synapses and increased dendritic arborization of pyramidal neurons occurring in the contralateral hemisphere are associated with the recovery of sensorimotor function following unilateral stroke in rats . Autoradiographic data show a role for the contralateral cortex in the behavioral outcome and maintenance of the recovered state of ischemic rats with unilateral filament MCA occlusion . PET and fMRI studies in rats show increased blood flow and metabolic activity in the contralateral hemisphere, which parallels functional recovery after stroke . The poor functional recovery of aged rats after stroke has been related to a reduced transcriptional activity in the contralateral hemisphere . Contrast-enhanced fMRI also showed the involvement of the contralateral somatosensory fields in brain reorganization in rats subjected to unilateral MCA occlusion . EEG power increase in the contralateral cortex in patients with unilateral ischemic stroke in the MCA territory was associated with negative prognosis of stroke .
An interesting finding was the observation of appearance of HVSs in the contralateral S1BF in the middle-aged rats by days 7–14 after stroke induction. HVSs are generalized EEG patterns typically seen in rats older than 6–8 months that are motionless during waking state [30, 55] and are thought to represent a distinct functional mode of thalamo-cortical circuits . The role of the thalamus in the recovery processes after ischemia is not clear. Data on changes in thalamus after unilateral ischemia or damage in the cortex are scare in preclinical models of stroke. Interestingly, two weeks after occlusion of MCA, the fMRI response to stimulation of the affected forelimb is shown to be absent in the ipsilateral thalamus although it can be detected in the ipsilesional cortex . Changes in neuroplasticity occurs in the thalamus, where retrograde neuronal degeneration has been found after cortical damage . However, to our knowledge there are no studies that examine how the age- and motionless-related rhythmical patterns like HVSs might be influenced by stroke. In our longitudinal EEG studies we observed high expression of HVSs in ipsilateral cortex one to eight months after Et-1 infusion. These findings are in line with reported enhanced rhythmogenic properties of the ipsilateral thalamocortical neurons with long delay (days, months) after photothrombotic focal cortical stroke in 7-8-month old rats . The functional significance of HVSs during quiet waking in rodents is not clear, as yet  and it is not known how they might influence the recovery process after stroke. Analyzing together the EEG data obtained from middle-aged rats monitored for 14 days after Et-1 infusion and EEG data from rats monitored for 8 months after Et-1 infusion, we showed that HVSs appeared first in the contralateral cortex (7–14 days) and later (after the first month) in the ipsilateral cortex. These findings lend credit to the hypothesis of the biphasic sequence of events after occlusion of MCA in rats (fMRI experiments): an initial reorganization in the contralesional hemisphere and a late recruitment of the ipsilesional periinfarct cortex . The roles of contralateral and ipsilesional cortical areas together with the thalamocortical circuits in the recovery process and plasticity after unilateral stroke-induced ischemia merit further investigation.