Intranasal Delivery Of Transforming Growth Factor-beta1 Is Neuroprotective Against Hippocampal Damage After Lithium-pilocarpine Induced Status Epilepticus

Objective: Intranasal administration provides a noninvasive and effective method for bypassing blood-brain barrier (BBB) to deliver TGFβ1 to the central nervous system (CNS) of status epilepticus (SE) rats. To investigate the potential neuroprotectional role of intranasal TGFβ1 against hippocampal damage after lithium-pilocarpine induced status epilepticus, and to explore the underlying mechanisms.Method: 60 Sprague-Dawley (SD) rats were randomly enrolled into the TGF group, Pilo group and the Control group. The lithium-pilocarpine induced SE was as the SE model. Diazepam (10 mg/kg i.p) was injected 50 min after the noset of SE to terminate seizue activity. Control animals were treated identically to the experimental group, but saline (0.9%) was given instead of pilocarpine. Recombinant human TGFβ1 was intranasally administered after SE cession immediately in TGF group. Meanwhile, rats in control group and pilocarpine group were treated identically with the same volume of saline. Rats were anesthetized and flush perfused transcardically 6 hours, 24 hours, 48 hours, and 72 hours after SE (n=5 for all time points). Then rats brains were carried out into sections in coronal. Morphological changes of hippocampal neurons were observed by hematoxylin-eosin (HE) staining. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method was applied to determine in situ apoptosis in the hippocampus. Fluoro-Jade B (FJB) stain dye was then used to visualize the dynamic changes of neuronal degeneration in the rat hippocampus in every group. Immunohistochemistry was conducted to detect the expression of Bcl-2, Bax and Caspase-3 in hippocampal neurons.Results: (1) Normal neurons were regularly aligned and the structure was intact including transparent cytoplasm, round or oval nuclei, chromatin distribution and clear nucleoli. While HE staining showed that the cell bodies were irregularly aligned and became shrunken with the shape of irregular triangle, some nuclei were shrinkage and had the shape of crescent, and The structure of hippocampal neurons is unclear at 6 h after SE, which presenting mainly in CA1, CA3 and DG of hippocampus. Neurons in the hippocampus were more intensely stained and the extent of morphological changes became gradually severe, while peaked at 72 h after SE in Pilo group. The extent of these phological changes peaked at 48 h, while the morphology and staining of neurons close to normal increasing at 72 h after SE in TGF group. Morever, intranasal TGFβ1 in TGF group markedly attenuated these phological changes in Pilo group at the time point of 24 h,48 h and 72 h after SE. (2) FJB positive cell was observed in both Pilo group and TGF group, presenting mainly in DG and CA1 of hippocampus, which appeared bright yellow-green against a dark background. No FJB positive cell was observed in the control group. FJB positive cells appeared at 6 h after SE and then increased gradually, while peaking at 72 h after SE in Pilo group. Meanwhile, FJB positive cells were observed at 6 h after SE and peaked at 48 h, while decreasing at 72 h after SE in TGF group. Moreover, intranasal TGFβ1 in TGF group significantly reduced the number of FJB-positive cells in Pilo group at 24 h (P < 0.05), 48 h (P < 0.05) and 72 h (P < 0.01), with no significant difference at 6 h between Pilo and TGF groups. Both Pilo and TGF groups had significantly higher expression of FJB-positive cells compared to the control (P < 0.01). (3) TUNEL staining showed that nuclear condensation with the shape of irregularity, which appeared brownish yellow ~ yellow. Chromatin intensely stained and nuclear fragmentation were examined under a high power microscope, which were consistent with apoptotic morphological changes. TUNEL positive cell was observed in both Pilo group and TGF group, presenting mainly in CA1 and CA3 of hippocampus, while no or few TUNEL positive cell was observed in the control group. TUNEL positive cells appeared at 6 h after SE and then increased gradually, while peaking at 72 h after SE in Pilo group. Meanwhile, TUNEL positive cells were observed at 6 h after SE and peaked at 48 h, while decreasing at 72 h after SE in TGF group. Moreover, intranasal TGFβ1 in TGF group significantly reduced the number of TUNEL-positive cells in Pilo group at 24 h (P < 0.05), 48 h (P < 0.05) and at 72 h (P < 0.01), with no significant difference at 6 h between Pilo and TGF groups. Both Pilo and TGF groups had significantly higher expression of TUNEL-positive cells compared to the control (P < 0.01). (4) Bcl-2 positive cells mainly expressed in the cytoplasmic or nuclear membrane, which appeared brownish yellow ~ brown. Bcl-2 positive cell was observed in both Pilo group and TGF group, presenting mainly in CA1 and CA3 of hippocampus, while no or few Bcl-2 positive cell was observed in the control group. Bcl-2 positive cells were observed at 6 h after SE and peaked at 48 h, while decreasing at 72 h after SE in Pilo group. Meanwhile, Bcl-2 positive cells appeared at 6 h after SE and peaked at 24 h, while decreasing persistently until 72 h after SE in TGF group. Moreover, intranasal TGFβ1 in TGF group significantly increased the number of Bcl-2-positive cells in Pilo group at 24 h (P < 0.01), 48 h (P < 0.05) and 72 h (P < 0.05), with no significant difference at 6 h between Pilo and TGF groups. Both Pilo and TGF groups had significantly higher expression of Bcl-2-positive cells compared to the control (P < 0.01). (5) Caspase-3 was mainly expressed in the cytoplasm or nucleus, which appeared brownish yellow or brown. Caspase-3 positive cell was observed in both Pilo group and TGF group, presenting mainly in CA1 and CA3 of hippocampus, while no or few Caspase-3 positive cell was observed in the control group. It showedessentially the same pattern as observed with the TUNEL labelling and a finding consistent with the results of the TUNEL assay. (6) Bax positive cells mainly expressed in the cytoplasmic or nuclear membrane, which appeared brown. Bax positive cell was observed in both Pilo group and TGF group, presenting mainly in CA1 and CA3 of hippocampus, while no or few Bax positive cell was observed in the control group. It showed essentially the same pattern as observed with the TUNEL labelling and a finding consistent with the results of the TUNEL assay.Conclusion: Intranasal delivery of Transforming growth factor-beta1 can significantly decrease the neurons undergoing degeneration and apoptotic death in rats hippocampus after SE and exert potential neuroprotective effect. Its underlying neuroprotective machanisms were likely to relate to up-regulating Bcl-2 expression and down-regulating Bax and Caspase-3 expression. Intranasal administration is a noninvasive and practical method for bypassing blood-brain barrier (BBB) to deliver drugs to the central nervous system (CNS), which may offer a promising strategy for treating Epilepsy, Alzheimer'disease and other CNS disorders.