Although rehabilitation is crucial for enhancing functional recovery if initiated early after stroke, it does not replace lost tissue. Transplanting multipotent neural stem cells is one method to replace the lost cells. In this work, we enhanced neurogenesis in the subventricular zone of mice by electric pulses and transplanted the stimulated cells into the infarct area of mice after mixing with a nutritional hyaluronic acid-based gel at day 14 post-stroke. Behaviorally, at 2 weeks after the treatment, the animals that received the transplanted cells performed significantly better in the Open field, Novel object, Hole board and Grooming tests and had a better score for the „time-to-feel” index in the adhesive tape test. At 28 days post-stroke, the infarct area was fully covered with a healthy-looking soft tissue filled in with cells expressing the neuroepithelial marker nestin and numerous cells expressing the transcription factor Mash1 (Ascl1), a marker of neural progenitor cells.
In previous work, we reported that enhancement of neurogenesis in the subventricular zone by electrical stimulation of post-stroke aged rats led to an improved functional recovery of spatial long-term memory (T-maze) but not on tasks requiring complex sensorimotor skills on the rotating pole [18]. However, the corpus callosum may act like a barrier thereby preventing the migration of neuronal precursor cells from the SVZ to the lesioned cortical area [5]. Therefore, we reasoned that by combining newly formed stem cells, derived from neurogenic stimulation of the SVZ, with a nutritional hydrogel which is then transplanted into the stroke cavity 2 weeks after the ischaemic event may enhance the odds of structural and behavioral recovery after stroke. Further, by administering the SVZ graft at two weeks after stroke, we avoided the detrimental effects of phagocytosis on the transplanted cells [23].
Improving behaviour, including ability to move, exploratory behaviour, cognition and memory are essential to any successful stroke therapy. Of these, we found that animals that received the SVZ graft performed better in variety of behavioral tests including Open field, Novel object, Hole board and Grooming tests. The Open field test provides clues on general ambulatory ability and anxiety-related emotional behaviors [24]. Likewise, in the open field test animals that received SVZ cells grafts increased their movement velocity by a factor of 2 as compared to controls, suggesting a better recovery in the muscle strength.
The object recognition test (ORT) is commonly used to asssess various aspects of learning and memory in mice and reflects the relative health of brain regions involved in short-term memory [25]. We found that the SVZ graft treatment led to an improvement in short-term memory reflecting a better preservation of this function in the brain of treated animals.
The hole board test can be used to assess exploratory, habituation, locomotor and anxiety behaviour. An increased number of head dippings in the treated animals may reflect a recovery in these functions which deteriorated pregressively in control animals. Finally,
a better score for the „time-to-feel” index in treated animals suggests a better recovery of sensory function in the adhesive tape test.
In our experimental approach we used ES to stimulate neurogenesis in the SVZ and increase the number of proliferating cells including neuronal precursors in the transplanted hydrogel. Indeed, the number of proliferating cells expressing BrdU, nestin, Mash1 and DCX was greatly increased after repeated stimulation. Following trnasplantation, nestin/BrdU double labeled cells as well as numerous Mash1-positive cells were detected at two weeks after transplantation. However, by immunohistochemistry, DCX-positive cells were not found in the transplanted gel suggesting that DCX-positive cells neither survived, nor differentiated into mature neurons after transplantation.
Understanding neural stem cell regulation is essential to improve our stem cell therapy for stroke for stroke [26]. Nestin and DCX are well known markers of cell proliferation in the SVZ [27]. In a recent study, it was shown that nestin is expandable in vitro and its expression persists in neurospheres grown in vitro after 7 days in culture derived from the subventricular zone of adult mice. Of note, DCX could not be detected in neurospheres at 7 days in culture confirming our results [19]. In previous work we found that DCX was robustly induced in the SVZ and hippocampus of young and aged rats subjected to cerebral ischemia. However, DCX did not reach the infarcted area and does not contribution to neuronal repair after stroke [6, 13, 28].
Neural and oligodendrocyte progenitor cells in the adult brain express Mash1 (also known as Ascl1), coding for a basic helix-loop-helix transcription factor. Proneural genes such as Ascl1 are known to promote cell cycle exit and neuronal differentiation when expressed in neural progenitor cells. Mash1 is another representative gene of the SVZ (Kim et al., 2011) that can be expanded in vitro and found to be capable of diferentiating into neurons when exposed to an appropriate environment [29, 30]. In this work we found that in the postnatal mouse brain Mash1 expression is seen in tubular thread-like structures similar in appearance to the structures seen in the SVZ after ES. Most importantly, in the transplanted animals, Mash1 expression survived for at least 2 wks in the lesional area. Thus, we found a striking parallel between nestin cell emanating from injured blood vessel in the ischemic area [5] and the tight association of Mash1 expression with Col IV expressed by blood vessels in the infarcted region of transplanted animals. Of note, much like the neuroepithelial marker Nestin, Mash1 is expressed during development in the hypothalamic neuroepithelium of mouse and is downregulated in the adult brain [22]. Indeed, in the CNS Mash1 mRNA is localized to the neuroepithelium that correspond to future functionally distinct areas of the brain [31].
During neurogenesis MASH1 expression is confined to mitotically active precursor cells being involved in the early stages of lineage commitment thereby upregulating NeuroD in neural precursor cells to direct terminal differentiation. However, Mash1 expression during development is not restricted to the brain. It has been also described in the development of adrenal medullary chromaffin cells, ganglion cells, thyroid parafollicular C cells, pulmonary neuroendocrine cells and even in small cell carcinomas [32, 33].
Quite surprinsingly, Mash1 expression has not been described in detail in brain injuries. It has been used, however, to convert NG2 glia into neurons in combination with two other transcription factors, Lmx1a, and Nurr1 to functionally mature neurons that integrate into existing brain circuitry after a lesion to the striatum in mice [34]. Similarly, Ascl1 in conjunction with Dlx2 has been used to genetically convert reactive glia into interneurons in a mouse model of epilepsy [35].