Fig 3.5: Neuroblasts within the lesion. DCX-positive cells (a) within the lesion (region with-out NeuN staining, circumscribed by dashed line in (b)), showing the typical morphology of mi-grating neuroblasts (cp (Martinez-Molina et al. 2011)). d – f show higher magnification pho-tomicrographs of the relocated neuroblasts. All images depict sagittal plane, orientation: left:
caudal, top: dorsal; scale bars: a – c: 500 µm; d – e: scale bars 50 µm.
Fig 3.6: DCX-positive neuroblasts found within the lesion. Both doses of endoN (L/E and L/
EHi) caused significantly (P < 0.001) more neuroblasts to migrate into the lesion than in le -sioned and sham-injected (L/C) animals. The doses did not result in different cell numbers.
the lateral ventricle of lesioned rats, significantly more DCX-positive neuroblasts were found within the lesioned tissue, compared to that of control animals.
3.5.1 Effect of endoN-Treatment
As revealed by the ex vivo experiment, endoN removed all detectable PSA-NCAM from free floating rat brain sections even at a lower concentration than used in vivo. There-fore, it was somewhat surprising to find PSA-NCAM in the RMS of endoN-treated ani-mals. However, PSA-NCAM was mostly detected in the SVZ and the RMS, whereas the surrounding tissue showed little PSA-NCAM immunoreactivity.
Although it is possible that the endoN applied only had an incomplete effect, we hy-pothesize that the effect of endoN has rather decayed and the PSA-NCAM immunoreac-tivity has partially recovered until the end of the experiments. This assumption is sup-ported by the lack of difference between the applied doses of endoN. In general, the en-zyme is reported to cleave PSA-NCAM completely for a prolonged time of at least 7 days after intracranial injection (Ono et al. 1994; Burgess et al. 2008; Battista and Rutishauser 2010). In a study on young mice endoN was injected into the lateral ventri-cle at PD1 (Seki and Rutishauser 1998). Here, PSA-NCAM was found to be partially re-expressed after 30 days and almost fully restored after 1.5 months. On the other hand, another study found re-occurring PSA-immunoreactivity in parts of the hippocampus of rats after electric stimulation as early as five days after a single injection of endoN into the ventricle (Pekcec et al. 2007).
In the present study we also evaluated the effects of endoN application on the structure of the RMS. Although our study was not opted for the RMS measurements, which re-sulted in a low power of these data, the measurements indicate that the enzymatic treat-ment led to a widening of the caudal and rostral RMS compared to controls. Further-more, the structure of the RMS of endoN-treated animals was less organized and the typical chains of neuroblasts were observed less frequently. These results go along with other studies in which endoN caused PSA-NCAM to become completely undetectable (Battista and Rutishauser 2010).
Taken together, the change in the RMS structure as well as the effect of endoN on mi-grating neuroblasts (see below), demonstrate the potency of the endoN injection in our experiments, although the effect on PSA-NCAM immunoreactivity was less pronounced than in other studies.
3.5.2 Effects of the excitotoxic lesion and endoN on migrating neuroblasts
Under physiological conditions neuroblasts are guided by chemoattractive and –repul-sive signals towards their target locations (reviewed in (Sun et al. 2010; Leong and Turnley 2011)). However, also damaged tissue in the brain attracts migrating lasts over a shorter distance. This has been described in studies of spontaneous neurob-last migration towards damaged tissue (Jin et al. 2003; Lee 2006; Kunze et al. 2015), but is also backed by our earlier work (Gundelach and Koch 2018), in which a known
chemoattractive substance, laminin, was used to redirect neuroblasts from the RMS. Af-ter the cells had left the artificial migration path, they kept on migrating in a now ran-domly oriented manner throughout the brain lesion.
Previous studies have demonstrated the importance of PSA-NCAM for the chain migra-tion of neuroblasts tangentially through the RMS towards the olfactory bulb, whereas the radial migration of single cells seems to be largely unaffected by the lack of PSA-NCAM (Ono et al. 1994; Hu et al. 1996). However, a more recent study found PSA overexpression to enhance the sensitivity of single neural precursor cells towards vari-ous migratory cues (Glaser et al. 2007).
We demonstrated that SVZ-derived cells, once they have left the RMS, migrate towards a brain lesion despite the previous removal of PSA-NCAM. Since the migration of indi-vidual cells rather parallels the radial migration of neuroblasts in the OB, it is not sur-prising that this mechanism remains functional. However, our results also show that the migratory cues released by the damaged tissue are capable of directing the neuroblasts over a distance of several millimeters. This finding verifies that the migration of neurob-lasts towards lesion sites is based on chemokines (Belmadani 2006; Robin et al. 2006;
Yan et al. 2007), rather than the PSA-regulated PI3K receptor (Glaser et al. 2007).
3.5.3 Fate of relocated cells
In our present study, no cells both immunoreactive for DCX and NeuN were found within the brain lesions. This stands in contrast to our previous work on relocated neu-roblasts in which a small number of cells showed morphological and immunological signs of differentiation (Gundelach and Koch 2018). It is possible that the relocated cells need more than the provided survival time of three weeks to migrate and differenti-ate. However, under physiological conditions most neuroblasts develop into OB-neu-rons within 15-30 days after proliferation (Petreanu and Alvarez-Buylla 2002). Interest-ingly, endoN-based removal of PSA from NCAM has been demonstrated to promote cell differentiation of neuroblastoma cells (Seidenfaden et al. 2003) as well as SVZ-neu-roblasts: After demyelination of the corpus callosum, endoN-treatment not only en-hances migration of SVZ cells towards the lesion, but also causes the cells to differenti-ate towards a oligodendroglial cell type (Decker et al. 2002). Furthermore, neuroblasts that do not leave the SVZ after endoN-treatment were found to differentiate towards a neuronal morphology and express tyrosine hydroxylase, resembling OB cells (Petridis et al. 2004). In vitro experiments revealed that this effect is cell-cell contact dependent and can be reduced by anti-NCAM antibodies. However, tyrosine hydroxylase expres-sion was not detected in the cultured cells (Petridis et al. 2004). Together these findings indicate that the fate of neuroblasts can be largely influenced by the interaction with the surrounding tissue. Physiologically, these influences on proliferating and migrating neu-roblasts are prevented by the polysialated NCAM within the cell membranes. Once the cells reach their position within the OB, PSA-NCAM is downregulated (Rousselot et al.
1995), thus a similar, contact dependent fate determination is conceivable. In our experi-ments a large volume of the surrounding tissue was lesioned, thus reducing the potential
of interaction between neuroblasts and intact nerve cells. Also the neuroblasts were found to spread within the lesion, which makes an interaction between these cells and an endoN-induced cell differentiation unlikely.
3.5.4 Potential for the development of a clinical application
The long term objective for the present study is to evaluate the endoN-induced neurob-lasts migration towards a brain lesion in the context of a clinical application after struc-tural brain damage in humans.
The general existence of postnatal neurogenesis in the human SVZ and hippocampus are well established (Curtis et al. 2011), however, there are differences between the ani-mal model and humans. The knowledge on human neurogenesis and neuroblast migra-tion is widely based on data from elderly subjects, since only post-mortem analysis is applicable. Furthermore, the widely used protein markers for developing neuronal cells can not always be interpreted unequivocally. This is not only the case for DCX-positive astrocytes (Kunze et al. 2015; Gundelach and Koch 2018), also markers for adult stem cells are expressed by a non-proliferative cell type (Gebara et al. 2016). Therefore, cells often need to be identified by the co-expression of multiple markers or by the cell mor-phology. These limitations make the transfer to the human brain especially challenging.
Recently, the persistence of neurogenesis throughout adulthood has been challenged (Sorrells et al. 2018) which has kindled a discussion about neurogenesis in the adult hu-man brain (Snyder 2018). Apart from the recent debate, it is known that the initial gen-eration of new neurons in the SVZ and hippocampus declines after infancy (Sanai et al.
2011; Bergmann et al. 2012). A similar, yet less pronounced decline has also been found in rodents (Kuhn et al. 1996; Tropepe et al. 1997; Shook et al. 2012). A better under-standing of this age related change might eventually provide methods to sustain neuro-genesis and neuroblast migration which in turn would provide new perspectives for clin-ical treatment of brain damage, based on endogenous neuroblasts.
In order to develop a viable treatment for structural brain damage, further conditions need to be fulfilled. Our data demonstrate the capability of SVZ-derived neuroblasts to migrate towards a brain lesion spontaneously, probably guided by chemoattractive cues (Fallon et al. 2000; Belmadani 2006; Robin et al. 2006; Yan et al. 2007; Courtès et al.
2011). It needs to be evaluated over what distances these cues affect neuroblasts after they were enabled to leave their predetermined path. Furthermore, the long term fate of re-located neuroblasts needs to be addressed. For example the inhibition of programmed cell death (Gascon et al. 2007; Kim et al. 2007) might increase the number of neurob-lasts within the lesion. Furthermore, we found no signs of newly differentiated neuronal cells within the lesions three weeks after the endoN-treatment, which means the previ-ously described differentiation stimulating effects of endoN (Petridis et al. 2004) do not show under the given experimental conditions. However, there are molecules known to promote differentiation of neuronal precursor cells, such as brain derived neurotrophic factor (Ahmed et al. 1995), epidermal growth factor (Reynolds and Weiss 1992), in-sulin-like growth factor-I (Arsenijevic and Weiss 1998), and leukemia inhibitory factor
(Memberg and Hall 1995). Application of these substances to relocated neuroblasts might enable their differentiation and integration into the surrounding neuronal tissue eventually.
3.5.5 Conclusion
The endoN-based technique enables migration of neuroblasts into a brain lesion and uti-lizes the migratory cues probably released by the lesion to guide the neuroblasts to their target. Therefore, it requires low surgical effort. However, the neuroblasts do not seem to differentiate within the lesion spontaneously. Furthermore, it is unclear to what extent neurogenesis declines after infancy in humans. It remains for future studies to determine if endoN might prove useful for the development of a future clinical treatment for struc-tural brain damage.
Acknowledgments
We thank Prof. Dr. Gerardy-Schahn and Prof. Dr. Hildebrandt for generously providing endoN and the antibody against PSA-NCAM as well as valuable information about the substances. Furthermore, we thank Maja Brandt for excellent technical assistance.