Institutionen för klinisk neurovetenskap / Department of Clinical Neuroscience
Abstract
Cerebrovascular disease and head trauma are among the leading causes of
death and disability. In addition to severe motor disturbances, ischemia
resulting from stroke or traumatic brain injury (TBI) can cause mild to
severe cognitive deficits. One-third of all patients with stroke show
hyperglycemia upon hospital admission, and the majority of these are
diabetic. The combination of diabetes and stroke was reported to worsen
recovery.
In agreement with clinical evidence, larger infarct sizes and
neurodegeneration were observed in rats following hyperglycemic brain
ischemia. The Goto-Kakizaki (GK) rat is a non-insulin-dependent,
spontaneously diabetic animal and, to our knowledge, few studies of
recovery from focal brain ischemia were performed in this rat strain. In
our laboratory, we have developed a model of experimental brain injury in
the Sprague-Dawley rat produced by transient, short-lasting (30 min)
unilateral extradural compression (EC) of the right sensorimotor cortex.
EC produces neurological manifestations (contralateral fore- and
hind-limb paresis) and selective neuronal death in the cortex,
hippocampus and thalamus, resembling clinical cortical stroke.
The aims of the present thesis were to (1) characterize motor and
cognitive deficits in both non-diabetic Wistar and diabetic GK rats
following EC, (2) to study compression induced-neurodegeneration and
potential correlations with the behavioural findings in non-diabetic
Wistar and diabetic GK rats, (3) to compare glucose levels and cerebral
blood flow (CBF) upon EC and reperfusion in both strains and (4) to
investigate strain differences in the expression of several antioxidant
and heme-degrading enzymes.
Recovery of motor and cognitive functions following EC were assessed with
the lever-press task (LPT) and locomotor activity (LMA) monitoring in a
novel environment, in parallel with the beam walking and the rotarod.
Neurodegeneration induced by EC was concomitantly characterized in
several brain regions by using Fluoro-Jade (FJ) as a marker of
neurodegeneration and GFAP as marker of reactive astrocytosis at 2, 5, 7,
10 and 14 days in Wistar rats and at 2, 7 and 14 days in GK rats.
Cortical CBF upon EC and during reperfusion was measured with
Laser-Doppler flowmetry. At 48 h post-EC, mRNA expression of
heme-degrading enzymes (HO-1, HO-2), biliverdin reductase (BVR),
superoxide dismutases (SOD-1, SOD-2), inflammatory and proapoptotic
markers (iNOS, TNFalpha, Bax) were compared between strains with real
time RT-PCR. HO-1 expression at 48 h post-EC was studied using
double-fluorescence immunohistochemistry for neurons (Fluorescent Nissl
staining), astrocytes (GFAP) and microglia (OX-42).
Locomotor and exploratory activities of compressed Wistar rats were
reduced, in parallel with hemiparesis, detected on the beam walking and
on the rotarod on day 1. The LMA parameters normalized on day 2, whereas
a phase of increased locomotor activity coupled with deficient
habituation to the environment was observed on day 3. Importantly, the
deficient habituation was no longer attributable to the motor
impairments. The learning of the LPT was delayed in naive-to-task Wistar
rats up to 10 days after EC. Fluoro-Jade/GFAP staining demonstrated a
consistent pattern of cortical, striatal and thalamic degeneration but
revealed variable degrees of degeneration in hippocampal areas. The
improvement in LPT performance of naïve-compressed rats was followed by a
reduction of damage in cortical associative areas. Additional
lesion-effects from damaged hippocampii may have overlapped in a minority
of subjects, while the subcortical lesions provoked by EC were unlikely
to explain the behavioural findings. GK but not control Wistar rats
showed a pronounced hyperglycemic response upon EC, a lower degree of
cortical CBF recovery during reperfusion, impaired behavioural
habituation to a novel environment on the first five days post-ischemia,
impaired learning of a LPT two weeks after EC and a higher degree of
neurodegeneration labelled by FJ in the cortex, hippocampus and thalamus
at virtually all time-points post-ischemia. Under basal conditions, GK
rats exhibited higher mRNA expression of heme degrading, antioxidant and
pro-inflammatory genes such as HO-1, iNOS and TNFalpha under basal
conditions. At 48 h post-ischemia, HO- 1 was one of the main upregulated
genes in the ipsilateral cortex of both diabetic and non-diabetic rats.
HO-1 secretion was localized in peri-lesional astrocytes and few
microglial cells.
Previous experience with the task and familiarity with the environment
appear to accelerate recovery from brain ischemia and may initiate
compensatory mechanisms at early stages of recovery, with emphasis on the
associative cortical areas. The GK rat consistently showed aggravated
hyperglycemia, worsened cortical reperfusion and longer-lasting
impairments of motor and cognitive functions encouraging further brain
injury studies in this rat strain. Counteracting oxidative stress caused
by heme degradation and neuroinflammation following normo- and
hyperglycemic brain ischemia may thus provide an effective therapy for
focal brain ischemia due to a potentially extended therapeutic window