Intravenous human umbilical cord blood transplantation for stroke: Impact on infarct volume and caspase-3-dependent cell death in spontaneously hypertensive rats

Transplantation of human umbilical cord blood cells (HUCBC) produces reliable behavioral and morphological improvements in animal models of stroke. However, the mechanisms of action still have not been fully elucidated. The aim of the present study is the evaluation of potential neuroprotective effects produced by HUCBC in terms of reduced infarct volume and caspase-3-dependent cell death. Permanent middle cerebral artery occlusion was induced in 90 spontaneously hypertensive rats. The animals were randomly assigned to the control group (n = 49) or the verum group (n = 41). The cell suspension (8 × 106 HUCBC per kilogram bodyweight) or vehicle solution was intravenously administered 24 h after stroke onset. Fifty subjects (n = 25/25) were sacrificed after 25, 48, 72 and 96 h, and brain specimens were removed for immunohistochemistry for MAP2, cleaved caspase-3 (casp3) and GFAP. Another 42 animals (n = 26/16) were sacrificed after 0, 6, 24, 36 and 48 h and their brains processed for quantitative PCR for casp3 and survivin. The infarct volume remained stable over the entire experimental period. However, cleaved casp3 activity increased significantly in the infarct border zone within the same time frame. Numerous cleaved casp3-positive cells were colocalized with the astrocytic marker GFAP, whereas cleavage of neuronal casp3 was observed rarely. Neither the infarct volume nor casp3 activity was significantly affected by cell transplantation. Delayed systemic transplantation of HUCBC failed to produce neuroprotective effects in a permanent stroke model using premorbid subjects

Intravenous human umbilical cord blood transplantation for stroke: Impact on infarct volume and caspase-3-dependent cell death in spontaneously hypertensive rats

Transplantation of human umbilical cord blood cells (HUCBC) produces reliable behavioral and morphological improvements in animal models of stroke. However, the mechanisms of action still have not been fully elucidated. The aim of the present study is the evaluation of potential neuroprotective effects produced by HUCBC in terms of reduced infarct volume and caspase-3-dependent cell death. Permanent middle cerebral artery occlusion was induced in 90 spontaneously hypertensive rats. The animals were randomly assigned to the control group (n = 49) or the verum group (n = 41). The cell suspension (8 × 106 HUCBC per kilogram bodyweight) or vehicle solution was intravenously administered 24 h after stroke onset. Fifty subjects (n = 25/25) were sacrificed after 25, 48, 72 and 96 h, and brain specimens were removed for immunohistochemistry for MAP2, cleaved caspase-3 (casp3) and GFAP. Another 42 animals (n = 26/16) were sacrificed after 0, 6, 24, 36 and 48 h and their brains processed for quantitative PCR for casp3 and survivin. The infarct volume remained stable over the entire experimental period. However, cleaved casp3 activity increased significantly in the infarct border zone within the same time frame. Numerous cleaved casp3-positive cells were colocalized with the astrocytic marker GFAP, whereas cleavage of neuronal casp3 was observed rarely. Neither the infarct volume nor casp3 activity was significantly affected by cell transplantation. Delayed systemic transplantation of HUCBC failed to produce neuroprotective effects in a permanent stroke model using premorbid subjects

Bone marrow cell transplantation time-dependently reverses G-CSF effects after stroke in hypertensive rats

Granulocyte colony-stimulating factor (G-CSF) is a hematopoietic cytokine and preclinically proven neuroprotectant. A potential reason for the clinical failure of G-CSF may be that relevant G-CSF effects such as the mobilization of mononuclear hematopoietic stem/progenitor cells from the bone marrow may take too long in humans (up to 9 days) to counter initial stroke consequences. Systemic transplantation of bone marrow mononuclear cells (BMMNCs) is feasible within a relatively short time after stroke onset and may provide an external resource of aforementioned stem/progenitor cells, thereby “bridging the gap” until G-CSF comes to full effect. Male spontaneously hypertensive rats (SHR) were randomly assigned into four groups after permanent middle cerebral artery occlusion (MCAO). Groups 1–3 received IP G-CSF treatment (50 μg/kg) for 5 days starting 1 h after stroke onset. Groups 2 and 3 also received 1.5 × 107/kg BMMNCs IV at 6 or 48 h following stroke, respectively. Group 4 received placebo treatment. Functional deficits (adhesive removal test), infarct volume, and edema (T2 TSE MRI) were repeatedly assessed for 1 month. Peripheral leukocyte counts and BMMNC biodistribution were analyzed by flow cytometry during the first week after stroke. G-CSF monotreatment reduced functional deficits (p 0.05 each). Surprisingly, BMMNC transplantation at 48 h abolished G-CSF effects. Early biodistribution studies (at 52 h after stroke onset) revealed splenic accumulation of granulocytes and BMMNCs as well as a granulocyte overload in the peripheral circulation and the brain (p < 0.05). Splenic accumulation of transplanted BMMNCs may have impaired peripheral granulocyte clearance. Subsequently, increased granulocyte numbers in the circulation and the poststroke brain prompted a proinflammatory bias of the innate immune system’s response to stroke, ultimately abolishing G-CSF effects. These surprising findings indicate that systemic effects of experimental stroke therapies need to be carefully considered when assessing the therapeutic potential of such novel approaches

Spontaneous white matter damage, cognitive decline, and neuroinflammation in middle-aged hypertensive rats: An animal model of early stage cerebral small vessel disease

Objectives Cerebral small vessel disease (cSVD) is one of the most prevalent neurological disorders. The progressive remodeling of brain microvessels due to arterial hypertension or other vascular risk factors causes subtle but constant cognitive decline and substantially increases the risk for stroke. Preliminary evidence suggests a contribution of the immune system to disease initiation and progression. Since most cSVD animal models are biased towards the hemorrhagic component of the disease1, a more detailed understanding is currently impaired by the unavailability of appropriate animal models. Here, we investigated the spontaneously hypertensive rat (SHR) as a possible model for early onset cSVD. Methods Male SHR and normotensive Wistar Kyoto rats (WKY, n=16 each, 11 weeks at enrolment) were used in this study. Animals were assigned to four experimental groups (Fig. 1). In group 1 (n=3/3), blood brain barrier (BBB) integrity was assessed by FITC-lectin and Evans Blue at 24 weeks. A brain tissue leukocyte profile of was obtained from group 2 (n=3/3) by fluorescence activated cell sorting (FACS) in week 35. In groups 3 and 4 (n=5/5 each), blood pressure was measured biweekly from week 12 to 22. Animals were further subjected to the novel object recognition (week 30) and Morris Water Maze (week 34) tests. Total brain, ventricle and corpus callosum (CC) volumes were determined by cerebral T2 MRI (3T) in week 35. Postmortem analyses included detailed cerebrospinal fluid, peripheral blood analysis by FACS and gene expression studies (laser microdissection and PCR), as well as detailed brain histology (neural cells, white matter density (Luxol Fast Blue), vessels and macro-/microglia). Results Blood pressure in SHR was significantly higher and increased over time (p<0.01 each). In contrast to agematched normotensive WKY, SHR exhibited non-spatial memory deficits (p<0.01). MRI showed brain atrophy (increased ventricle volumes and decreased CC/brain volumes; p<0.01). An increased myelin index, indicating myelin loss in SHR (p<0.01; Fig. 2). Histological analyses confirmed white matter demyelination and unveiled a circumscribed BBB dysfunction in conjunction with micro- and macrogliosis in deep cortical regions (DCR; p<0.05 or below; Fig. 3). FACS and histological analyses further revealed substantial disparities in cerebral CD45high leukocyte counts and distribution patterns between SHR and WKY. SHR showed lower T cells counts in the choroid plexus and meningeal spaces as well as decreased interleukin- 10 levels in the cerebrospinal fluid (p<0.05 or lower). Moreover, both T and NK cells were significantly augmented in the SHR brain microvasculature. Conclusions Our results indicate that SHR share behavioral and neuropathological characteristics with human cSVD patients and further undergird the relevance of immune responses for the initiation and progression of cSVD2.Fig. 2: SHR exhibited increased ventricle (A), decreased CC and brain volumes (B, C), and a higher myelin index (D) indicating white matter loss