Exploring the neuroprotective effects of modafinil in a marmoset Parkinson model with immunohistochemistry, magnetic resonance imaging and spectroscopy.

Neuroprotective therapeutics stop or slow down the degeneration process in animal models of Parkinson's disease (PD). Neuronal survival in PD animal models is often measured by immunohistochemistry. However, dynamic changes in the pathology of the brain cannot be explored with this technique. Application of proton magnetic resonance (MR) imaging (MRI) and spectroscopy (MRS) can cover this lacuna as these techniques are non-invasive and can be repeated over time in the same animal. Therefore, the sensitivity of both techniques to measure changes in PD-pathology was explored in an experiment studying the neuroprotective effects of the vigilance enhancer modafinil in a marmoset PD model. Eleven marmoset monkeys were treated with the neurotoxin 1-methyl-1,2,3,6-tetrahydropyridine (MPTP). Six of these 11 animals, simultaneously, received a daily oral dose of modafinil (100 mg/kg) and five received vehicle for 27 days. MR experiments were performed at baseline and 1 and 3.5 weeks after the MPTP intoxication period after which brains were analyzed with immunohistochemistry. Tyrosine hydroxylase immunoreactive (TH-IR) staining of dopamine neurons of the substantia nigra pars compacta confirmed that modafinil was able to partially prevent the MPTP-induced neuronal damage. In MRS, N-acetylaspartate (NAA)/phosphocreatine (tCR) ratios confirmed the protective effect indicating that this is a sensitive measure to detect neuroprotection in the MPTP marmoset model. Furthermore, the number of TH-IR positive neurons and the NAA/tCR ratio were significantly correlated to behavioral observations indicating that the changes measured in the brain are also reflected in the behavior and vice versa.

Blood-brain barrier permeability and monocyte infiltration in experimental allergic encephalomyelitis: a quantitative MRI study.

Enhanced cerebrovascular permeability and cellular infiltration mark the onset of early multiple sclerosis lesions. So far, the precise sequence of these events and their role in lesion formation and disease progression remain unknown. Here we provide quantitative evidence that blood-brain barrier leakage is an early event and precedes massive cellular infiltration in the development of acute experimental allergic encephalomyelitis (EAE), the animal correlate of multiple sclerosis. Cerebrovascular leakage and monocytes infiltrates were separately monitored by quantitative in vivo MRI during the course of the disease. Magnetic resonance enhancement of the contrast agent gadolinium diethylenetriaminepentaacetate (Gd-DTPA), reflecting vascular leakage, occurred concomitantly with the onset of neurological signs and was already at a maximal level at this stage of the disease. Immunohistochemical analysis also confirmed the presence of the serum-derived proteins such as fibrinogen around the brain vessels early in the disease, whereas no cellular infiltrates could be detected. MRI further demonstrated that Gd-DTPA leakage clearly preceded monocyte infiltration as imaged by the contrast agent based on ultra small particles of iron oxide (USPIO), which was maximal only during full-blown EAE. Ultrastructural and immunohistochemical investigation revealed that USPIOs were present in newly infiltrated macrophages within the inflammatory lesions. To validate the use of USPIOs as a non-invasive tool to evaluate therapeutic strategies, EAE animals were treated with the immunomodulator 3-hydroxy-3-methylglutaryl Coenzyme A reductase inhibitor, lovastatin, which ameliorated clinical scores. MRI showed that the USPIO load in the brain was significantly diminished in lovastatin-treated animals. Data indicate that cerebrovascular leakage and monocytic trafficking into the brain are two distinct processes in the development of inflammatory lesions during multiple sclerosis, which can be monitored on-line with MRI using USPIOs and Gd-DTPA as contrast agents. These studies also implicate that USPIOs are a valuable tool to visualize monocyte infiltration in vivo and quantitatively assess the efficacy of new therapeutics like lovastatin.

Losartan versus enalapril on cerebral edema and proteinuria in stroke-prone hypertensive rats.

Stroke-prone spontaneously hypertensive rats (SHRSP), subjected to high NaCl, show severe hypertension, organ damage, and early death. Preventive treatment with angiotensin II type 1 (AT1) receptor antagonists is known to be effective. Previously, we found that angiotensin converting enzyme (ACE) inhibition could reduce cerebral edema when treatment was started after manifestation of either proteinuria or cerebral edema. In this study AT1 receptor blockade was started at the same time points to evaluate whether this had an effect superior to ACE inhibition. SHRSP drank 1% NaCl. Group 1 served as controls. Group 2 and 3 rats were started on losartan and enalapril after proteinuria exceeded 40 mg/day. Group 4 and 5 rats were started on losartan and enalapril after the first observation of cerebral edema with T2-weighted magnetic resonance imaging scans. In controls, median survival was 54 days (range, 35 to 80 days) after the start of salt loading. With early-onset losartan and enalapril, survival increased to 305 days (range, 184 to 422 days) and 320 days (range, 134 to 368 days) (both P < .01 v group 1). Cerebral edema formation was prevented in all but two rats, one from each treatment modality. Development of proteinuria was markedly reduced. With late-onset treatment with losartan and enalapril, survival was 290 days (range, 120 to 367 days) and 264 days (range, 154 to 319 days) (both P < .01). Both losartan and enalapril decreased cerebral edema to baseline levels. Ultimately cerebral edema reoccurred, despite continued treatment, in 75% of the rats. Systolic blood pressure did not decrease after losartan treatment, but, similarly to early-onset treatment, decreased transiently after enalapril treatment. Cerebral edema and proteinuria were prevented and reduced in SHRSP treated with either an AT1 receptor antagonist or an ACE inhibitor. Survival was markedly and similarly prolonged by both treatments, whether initiated directly before or after development of cerebral edema. In rats where treatment was initiated after manifestation of cerebral edema, both cerebral edema and proteinuria reappeared despite continued treatment. Apparently, when hypertension is sustained, reappearance of target organ damage may not be entirely dependent on angiotensin.

MRI-based quantification of cerebral edema in individual SHRSP rats using averaged criteria determined before the occurrence of edema.

This study evaluated whether it is possible with T2-weighted MRI to quantitatively relate image-outcome in a single, individual case with that of a standardized control group that did not show these pathologies. An animal model of hypertension-related cerebral damage, the salt-loaded, stroke-prone spontaneously hypertensive rat (SHRSP), was applied. Very similar values for cerebral edema were found when using either the individual or the averaged threshold. The values were positively correlated with each, as edema(averaged threshold) = 0.12 + 0.99 x edema(individual threshold) (Pearsons coefficient = 0.99, p < 0.0001). This line was virtually congruent with the line of identity. Thus, by determination of the averaged threshold in the healthy salt-loaded SHRSP, a parameter was obtained to calculate cerebral edema with the specifically used T2-weighted MRI protocol, in any rat.

Early-onset but not late-onset endothelin-A-receptor blockade can modulate hypertension, cerebral edema, and proteinuria in stroke-prone hypertensive rats.

-The ability of endothelin receptor blockade to prevent and to treat established cerebral and renal injury was explored in salt-loaded stroke-prone spontaneously hypertensive rats (SHRSP) with the endothelin receptor subtype-A antagonist A127722. SHRSP were subjected to 1% NaCl intake. The start of treatment with A127722 (35 and 70 mg. kg-1. d-1, respectively) was either synchronized with salt loading or initiated after the first observation of cerebral edema with T2-weighted magnetic resonance imaging. In untreated control animals median survival was 54 days (range, 32 to 80 days) after the start of salt loading. Early-onset A127722 treatment increased median survival to 233 days (range, 92 to 407 days; P<0.05 versus controls) with 35 mg/kg and to 124 days (range, 97 to 169 days; P<0.05 versus control) with 70 mg/kg. The development of cerebral edema was prevented, and systolic blood pressure and proteinuria were dose-dependently reduced. However, all rats in the 70-mg/kg treatment group developed hemorrhages in the basal ganglia shortly before death. Late-onset A127722 treatment failed to affect survival, systolic blood pressure, or proteinuria. Nevertheless, cerebral edema was reduced but not as well as in early-onset treatment. Development of hypertension, cerebral edema, and proteinuria was prevented in SHRSP when A127722 treatment was initiated at the start of salt-loading. However, A127722 treatment did not prolong survival in SHRSP with cerebral edema. This suggests that in SHRSP the endothelin A receptor participates actively in the development of increased blood pressure and initiation of organ damage but participates minimally in established malignant hypertension and progression of target-organ damage.

Enalapril prevents imminent and reduces manifest cerebral edema in stroke-prone hypertensive rats.

BACKGROUND AND PURPOSE: Stroke-prone spontaneously hypertensive rats (SHRSP), subjected to high NaCl intake, show severe hypertension, organ damage, and early death. Preventive treatment with an angiotensin-converting enzyme (ACE) inhibitor is known to reduce mortality. Previously we found that proteinuria always precedes cerebral edema in SHRSP. Hence, in this study ACE inhibition was started later, ie, directly after manifestation of either proteinuria or cerebral edema. METHODS: SHRSP were subjected to 1% NaCl intake. Group 1 served as a control. In group 2 early-onset treatment with the ACE inhibitor enalapril was initiated after proteinuria was >40 mg/d. In group 3 late-onset ACE inhibition was started after the first observation of cerebral edema with T2-weighted MRI. Cerebral edema was expressed as the percentage of pixels with an intensity above a defined threshold. RESULTS: In controls median survival was 54 days (range, 32 to 80 days) after start of salt loading. The terminal level of cerebral edema was 19.0+/-3.0%. Under early-onset enalapril, median survival increased to 320 days (range, 134 to 368 days; P<0.01 versus group 1). Cerebral edema was prevented in all but 1 rat. Systolic blood pressure was slightly and transiently reduced at day 14. Proteinuria was markedly reduced (52+/-7 versus 190+/-46 mg/d in group 1 at day 7; P<0.05). Under late-onset enalapril, median survival was 264 days (range, 154 to 319 days; P<0.01 versus group 1). Cerebral edema decreased to baseline levels (9.6+/-2.9 at day 0 to 3.4+/-0.5% at day 3; (P<0.05). Ultimately cerebral edema reoccurred in 6 of the 8 rats. SBP decreased slightly at day 7 only. Proteinuria decreased from 283+/-27 at day 0 to 116+/-22 mg/d at day 7 (P<0.05). Complete remission of the original locus of cerebral edema was confirmed histologically. CONCLUSIONS: In SHRSP with proteinuria, treatment with an ACE inhibitor both prevented the development of cerebral edema and reduced manifest cerebral edema and proteinuria. Survival was markedly prolonged. These findings support the use of ACE inhibition for treatment in hypertensive encephalopathy.

Proteinuria precedes cerebral edema in stroke-prone rats: a magnetic resonance imaging study.

BACKGROUND AND PURPOSE: Stroke-prone spontaneously hypertensive rats (SHRSP) subjected to high sodium intake develop severe hypertension, cerebral edema, and proteinuria, culminating in organ damage and early death. MRI, which can be applied serially, provides the unique opportunity to study temporal and quantitative relations between these changes and whether diminution of sodium intake can attenuate established cerebral edema. METHODS: SHRSP were subjected to 1% NaCl in drinking water. Cerebral MRI, proteinuria and systolic blood pressure (SBP) were measured serially. After detection of cerebral edema (T2-weighted MRI), 6 rats were killed for histology, to confirm the diagnosis of cerebral edema. The others were followed up for 7 more days while salt loading was continued (n = 10, group 1) or after sodium intake was normalized (n = 7, group 2). RESULTS: SHRSP invariably developed cerebral edema in 30 days (range, 8 to 54 days). At this point neurological signs were absent in 16 of 23 rats. SBP rose until 1 week before detection of cerebral edema, and then stabilized at approximately 265 mm Hg. Proteinuria invariably preceded cerebral edema, with a concentration exceeding 40 mg/d predicting development of cerebral edema in 9 days (range, 3 to 15 days). There was linear correlation (R=.62, P<.0001) between proteinuria and cerebral edema (pixels with an intensity above a defined threshold). Rats in group 1 showed an increase in cerebral edema (from 5.8+/-1.1% to 12.5+/-2.8%; P<.05), and proteinuria remained high (from 305+/-44 to 338+/-29 mg/d); and 2 died spontaneously. Rats in group 2 showed no significant change in edema (from 4.9+/-0.5% to 6.9+/-1.3%) but a marked fall in proteinuria (from 294+/-24 to 119+/-10 mg/d; P<.05), both significantly different from group 1 (P<.05); all survived. SBP remained unaltered in both groups. CONCLUSIONS: Our data establish MRI as a sensitive method for detection of cerebral edema, often prior to neurological signs, in SHRSP. Proteinuria predicts cerebral edema, and these two variables, both obtained noninvasively, are quantitatively related. Moreover, in SHRSP normalizing sodium intake after salt loading attenuates development of cerebral edema and reduces proteinuria.