The major finding of this study is that intranasal administration of TGF-β1 reduces infarct volume, improves functional recovery and enhances neurogenesis in the adult mouse SVZ after transient focal ischemia.
Because the delayed administration of neuroprotective agents following ischemia is usually ineffective , we adopted early post-ischemia intranasal treatment in this study. Our data showed that there is a significant reduction in infarct volume in mice treated with TGF-β1, which is consistent with previous findings . TUNEL labeling indicated that ischemia induced DNA degradation was also decreased after intranasal administration of TGF-β1. Simultaneously, TGF-β1-treated animals exhibited progressive improvement on the NSS test compared with controls. All these support the hypothesis of a neuroprotective role for intranasal TGF-β1 in stroke. In addition to its function as a neuroprotective agent, TGF-β1 is a key regulator in development and cell cycle control [8, 25], suggesting that the functional recovery after stroke may be mediated by some other mechanisms. To our knowledge, this is the first time that the effect of intranasal TGF-β1 on neuroprotection and ischemia-induced neurogenesis in the SVZ was evaluated.
Adult neurogenesis consists of one or more of the following processes, including proliferation, survival, migration and differentiation . It is known that the number of BrdU immunoreactive cells in the SVZ increased with a peak at 7 days after ischemia, and then decreased gradually . The loss of BrdU-positive cells may be attributed to dilution of BrdU, apoptosis, and cell migration. In the present study, we demonstrated that intranasal treatment with TGF-β1 significantly increased BrdU-labeled cells both in the SVZ and striatum ipsilateral to the ischemia at each time point from 7 days after stroke. Despite the subsequent dilution of BrdU-labeling due to continued division or death of labeled cells, we also observed an increase in BrdU-positive cells at day 21 and day 28 after MCAO. Apart from enhancing survival of newborn cells, the increased number of BrdU-labeled cells in the injured striatum of TGF-β1-treated animals may result from an accelerated migration of nascent neurons from SVZ. Different from ICV administration, TGF-β1 may localize high concentration in the striatum  rather than adjacent SVZ after intranasal administration, and lead to enhanced migration from SVZ into ischemic region. As expected, double immunostaining showed that after treatment with TGF-β1, most BrdU-labeled cells were co-labeled with DCX, a microtubule-associated protein expressed in migrating neuroblasts, which confirmed the previous assumption. Furthermore, we found an increased number of BrdU/NeuN double-labeled cells in the SVZ and affected striatum of TGF-β1 treated group at the anaphase after stroke, whereas only a few cells were GFAP positive, which suggest that intranasal administration of TGF-β1 also promoted progenitor's differentiation towards a neuronal lineage. Taken together, above findings indicate that stroke-induced neurogenensis is facilitated after intranasal administration of TGF-β1.
Endogenous TGF-β1 is distributed in the proliferative zone, and its two receptors TβRI, TβRII are expressed by migrating neurons and radial glia . Recently, we reported that intranasal administration of TGF-β1 may exert its biological effects by regulating gene expression of TβRI and TβRII, but did not affect mRNA level of TGF-β1 itself, suggesting that the enhanced neurogenesis by TGF-β1 might be mediated through its receptors . It is known that neurogenesis is linked to the cell cycle, and neural stem cells may assume their particular neuronal or glial fates by exiting the cell cycle . In addition, modification of molecular morphogens and signals in the microenvironment of developing brain affects stem cell survival and differentiation . TGF-β1 promotes exit from the cell cycle exit by upregulating the expression of the cell cycle protein, p21 . Exogenous administration of TGF-β1 may interact with the endogenous system , upregulate cell adhesion molecule (CAM) expression , increase the potency of other neurotrophic factors involved in neurogenesis , modulate their action, and affect the signaling of classic neurotrophins , and thus enhance stroke-induced neurogenesis. The exact mechamisms for the effects of TGF-β1 on neurogenesis are still remain to be proven.
In concert with our results, a recent report showed that TGF-β1 also increased neurogenesis both in the hippocampal dentate gyrus of the adrenalectomized rats and in neural stem cells cultures even at physiological concentrations . Interestingly, some data from in vivo and/or in vitro studies indicate an adverse role of TGF-β1 in regulating neurogenesis. Wachs et al. reported that TGF-β1 induced a long-term inhibition of neurogenesis in the lateral ventricular wall and the dentate gyrus after 7 days of ICV infusion to adult female Fischer-344 rats . Buckwalter et al. observed that chronic overproduction of TGF-β1 also inhibited age-related neurogenesis in the hippocampus of aged transgenic mice . These discrepancies may be related to the dose of TGF-β1. In contrast to other neurotrophins, TGF-β1 has been shown to produce a marked effect in a concentration-dependent manner. In developing cortex, cell migration was promoted by TGF-β1 at low concentrations whereas at high concentrations it impaired migration . The decision of whether a community of progenitors undergoes predominantly neurogenesis or apoptosis is dependent on the concentration of TGF-β1 . It is likely that the effect of TGF-β1 on neurogenesis is also dose-dependent, related to the concentration achieved in the brain and also to the period of time that it remains elevated.
As a non-invasive method which bypasses the BBB, intranasal administration is an alternative drug delivery strategy for targeting the brain . Using this method, neuropeptides can be delivered to the brain directly from the nasal cavity for the time and treatment period needed. Two possible mechanisms shown to directly deliver drugs from the nasal mucosa to the brain along the neural pathway are the extracellular and the intracellular routes. Extracelluar transport along perineuronal and perivascular channels has been proposed to explain delivery of drugs to the brain within minutes [5, 6], whereas intracellular delivery requiring internalization of the drug within the neurons followed by axonal transport takes more than a few hours for drug distribution within the brain . We previously reported that TGF-β1 concentrations were significantly increased in several brain regions within 30 minutes after intranasal administration, while no increase was detected in the plasma and peripheral organs, suggesting that intranasal TGF-β1 is mainly transported into the CNS via extracellular neuronal pathways. Moreover, it was observed that the concentration of TGF-β1 following nasal delivery was sustained for at least 6 hours in some brain regions, such as striatum, thalamus, hippocampus and cortex, suggesting long-term stability of TGF-β1 tissue concentration when given via the nasal route . Thus, intranasal administration of TGF-β1 represents a promising modality for facilitating neuroprotection, neurogenesis and recovery of function after stroke. Whether direct neuroprotection, neurogenesis, or both contribute to the reduction of infarct volume and the improvement in neurological function requires future investigation.