Deepu R. Pillai

Temozolomide Preferentially Depletes Cancer Stem Cells in Glioblastoma

The prognosis of patients suffering from glioblastoma (GBM) is dismal despite multimodal therapy. Although chemotherapy with temozolomide may contain tumor growth for some months, invariable tumor recurrence suggests that cancer stem cells (CSC) maintaining these tumors persist. We have therefore investigated the effect of temozolomide on CD133(+) and CD133(-) GBM CSC lines. Although differentiated tumor cells constituting the bulk of all tumor cells were resistant to the cytotoxic effects of the substance, temozolomide induced a dose- and time-dependent decline of the stem cell subpopulation. Incubation with sublethal concentrations of temozolomide for 2 days completely depleted clonogenic tumor cells in vitro and substantially reduced tumorigenicity in vivo. In O(6)-methylguanine-DNA-methyltransferase (MGMT)-expressing CSC lines, this effect occurred at 10-fold higher doses compared with MGMT-negative CSC lines. Thus, temozolomide concentrations that are reached in patients were only sufficient to completely eliminate CSC in vitro from MGMT-negative but not from MGMT-positive tumors. Accordingly, our data strongly suggest that optimized temozolomide-based chemotherapeutic protocols might substantially improve the elimination of GBM stem cells and consequently prolong the survival of patients.

Cerebral ischemia-reperfusion injury in rats--a 3 T MRI study on biphasic blood-brain barrier opening and the dynamics of edema formation.

Serial magnetic resonance imaging (MRI) was performed to investigate the temporal and spatial relationship between the biphasic nature of blood–brain barrier (BBB) opening and, in parallel, edema formation after ischemia–reperfusion (I/R) injury in rats. T2-weighted imaging combined with T2-relaxometry, mainly for edema assessment, was performed at 1 h after ischemia, after reperfusion, and at 4, 24 and 48 h after reperfusion. T1-weighted imaging was performed before and after gadolinium contrast at the last three time points to assess BBB integrity. The biphasic course of BBB opening with a significant reduction in BBB permeability at 24 h after reperfusion, associated with a progressive expansion of leaky BBB volume, was accompanied by a peak ipsilateral edema formation. In addition, at 4 h after reperfusion, edema formation could also be detected at the contralateral striatum as determined by the elevated T2-values that persisted to varying degrees, indicative of widespread effects of I/R injury. The observations of this study may indicate a dynamic temporal shift in the mechanisms responsible for biphasic BBB permeability changes, with complex relations to edema formation. Stroke therapy aimed at vasogenic edema and drug delivery for neuroprotection may also be guided according to the functional status of the BBB, and these findings have to be confirmed in human stroke.

Neurovascular protection by targeting early blood–brain barrier disruption with neurotrophic factors after ischemia–reperfusion in rats

The ‘new penumbra’ concept imbues the transition between injury and repair at the neurovascular unit with profound implications for selecting the appropriate type and timing of neuroprotective interventions. In this conceptual study, we investigated the protective effects of pigment epithelium-derived factor (PEDF) and compared them with the properties of epidermal growth factor (EGF) in a rat model of ischemia–reperfusion injury. We initiated a delayed intervention 3 hours after reperfusion using equimolar amounts of PEDF and EGF. These agents were then administered intravenously for 4 hours following reperfusion after 1 hour of focal ischemia. Magnetic resonance imaging indices were characterized, and imaging was performed at multiple time points post reperfusion. PEDF and EGF reduced lesion volumes at all time points as observed on T2-weighted images (T2-LVs). In addition PEDF selectively attenuated lesion volume expansion at 48 hours after reperfusion and persistently modulated blood–brain barrier (BBB) permeability at all time points. Intervention with peptides is suspected to cause edema formation at distant regions. The observed T2-LV reduction and BBB modulation by these trophic factors is probably mediated through a number of diverse mechanisms. A thorough evaluation of neurotrophins is still necessary to determine their time-dependent contributions against injury and their modulatory effects on repair after stroke.

In vivo high-resolution imaging of the injured rat spinal cord using a 3.0T clinical MR scanner

To investigate the feasibility of obtaining high‐resolution MR images for the detection of pathological changes occurring in the injured rat spinal cord with a routine clinical 3.0T imaging system.

Delayed histochemical alterations within the neurovascular unit due to transient focal cerebral ischemia and experimental treatment with neurotrophic factors

Current stroke therapy is focused on recanalizing strategies, but neuroprotective co-treatments are still lacking. Modern concepts of the ischemia-affected neurovascular unit (NVU) and surrounding penumbra emphasize the complexity during the transition from initial damaging to regenerative processes. While early treatment with neurotrophic factors was shown to result in lesion size reduction and blood-brain barrier (BBB) stabilization, cellular consequences from these treatments are poorly understood. This study explored delayed cellular responses not only to ischemic stroke, but also to an early treatment with neurotrophic factors. Rats underwent 60 minutes of focal cerebral ischemia. Fluorescence labeling was applied to sections from brains perfused 7 days after ischemia. Analyses focused on NVU constituents including the vasculature, astrocytes and microglia in the ischemic striatum, the border zone and the contralateral hemisphere. In addition to histochemical signs of BBB breakdown, a strong up-regulation of collagen IV and microglia activation occurred within the ischemic core with simultaneous degradation of astrocytes and their endfeet. Activated astroglia were mainly depicted at the border zone in terms of a glial scar formation. Early treatment with pigment epithelium-derived factor (PEDF) resulted in an attenuation of the usually up-regulated collagen IV-immunoreactivity. However, glial activation was not influenced by treatment with PEDF or the epidermal growth factor (EGF). In conclusion, these data on ischemia-induced cellular reactions within the NVU might help to develop treatments addressing the transition from injury towards regeneration. Thereby, the integrity of the vasculature in close relation to neighboring structures like astrocytes appears as a promising target.