Tat-mediated Delivery Of Neuroglobin Protect Against Focal Cerebral Ischemia In Mice

Background and ObjectivesAcute stroke is one of the leading causes of death and long-term disability worldwide. So far, neuroprotection in patients with stroke has been unsuccessful and there is an urgent need for new therapeutic approaches. Neuroglobin (Ngb) is a newly identified globin in the vertebrate brain and is suggested to be neuroprotective against brain hypoxic/ischemic injury. However, the impermeability of the large molecule Ngb to the blood-brain barrier limits its application in the brain injury. Thus, an efficient and safe method for delivering Ngb across the BBB is needed. Recently, the 11-amino acid human immunodeficiency virus TAT protein transduction domain has been successfully used to deliver macromolecules into the brain. It was shown that TAT-neuroglobin (TAT-Ngb) generated by fusing Ngb with the PTD of HIV-TAT can efficiently transduce rat cortical neurons and human islets in culture, and protect cultures from hypoxia in vitro. The in vitro studies have suggested that the transduction of TAT-Ngb may be a promising approach for stroke treatment. However, until now, the effect of the TAT-Ngb fusion protein has not been demonstrated in an in vivo model. The present study evaluated whether TAT-neuroglobin (TAT-Ngb) fusion protein could cross the blood-brain barrier and protect the brain from cerebral ischemia. Methods1. The pET-TAT-Ngb and pET-Ngb expression vectors were constructed using gene recombinant technology, then the TAT-Ngb and Ngb fusion proteins were generated in Escherichia coli BL21(DE3). The fusion proteins purified by a Ni-NTA resin column were verified by SDS/PAGE, Coomassie Brilliant Blue staining and Western Blot analysis.2. The mice were injected intravenously with TAT-Ngb or Ngb and sacrificed 1 or 4 h after injection. Western Blot and immunofluorescence staining were applied to the cerebral hemisphere in order to observe the transduction of TAT-Ngb.3. The mice were anesthetized with isoflurane via a facemask and subjected to transient focal cerebral ischemia (30 min or 2 h) by middle cerebral artery occlusion (MCAO) with a modification of intraluminal filament technique. Regional cerebral blood flow, blood pressure and arterial blood gas were analyzed before and after MCAO. The mice were examined for neurological deficits and then decapitated. Brain injury in the brain coronal sections were respectively assessed by HE staining, Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining and cresyl violet staining.4. Mice treated with MCAO for 2 h followed by reperfusion was divided into sub-groups and was injected intravenously with TAT-Ngb, Ngb, and saline respectively before MCAO or immediately after reperfusion. At 24 h after MCAO, the mice were examined for neurological deficits and then decapitated. The cerebral infarct volume were determined by Triphenyltetrazolium chloride staining.5. Mice treated with MCAO for 30 min followed by reperfusion was divided into sub-groups and was injected intravenously with TAT-Ngb, Ngb, and saline respectively immediately after reperfusion. At 72 h after MCAO, the mice were examined for neurological deficits and then decapitated. Apoptosis and viable neurons in the brain coronal sections were respectively assessed by TUNEL staining and cresyl violet staining. TUNEL-positive cells and viable neurons in the 6 striatal regions of interest were quantified using light microscopy and ImageJ 1.42q.Results1. The recombinant plasmids pET-TAT-Ngb and pET-Ngb were verified by restriction enzyme analysis and the automated DNA sequencing. The purified fusion proteins were identified using Western Blot analysis with anti-His-tag and anti-Ngb antibodies.2. The TAT-Ngb fusion protein was efficiently delivered into the mouse brain tissue by intravenous injection as demonstrated by immunohistochemistry and Western Blot. but Ngb without TAT did not achieve detectable protein transduction.3. At 24 h after the 2-h MCAO treatment, the mice developed reproducible infarcts involving the cerebral cortex and the striatum, and the neurological deficit scores is 2.67±0.822. At 72 h after the 30 min MCAO treatment, the mice developed selective neuronal injury in the striatum, and none of the mice showed residual neurological deficit. There were no differences (p > 0.05) in mean arterial blood pressure, PaO2, PaCO2, pH, rectal temperature, or body weight between the different animal groups.4. Compared with Ngb- or saline-treated group, the group with TAT-Ngb treated 2 h before MCAO had significant less brain infarct volume and better neurologic outcomes (p < 0.05). Furthermore, in the group treated with TAT-Ngb after reperfusion, there was also a trend towards a reduction in infarct volume and neurological deficits, but it did not reach statistical significance.5. Compared with the saline-treated group, the number of TUNEL-positive cells was significantly (p < 0.01) reduced and neuronal viability was significantly (p < 0.01) increased in the group treated with TAT-Ngb after reperfusion but not in the Ngb-treated group.Conclusion1. Purified TAT-Ngb and Ngb fusion proteins were generated successfully using recombinant technology,2. The TAT-Ngb protein was able to cross the BBB after intravenous injection and enter neuronal cells3. The ransient focal cerebral ischemia mice model made by MCAO with a modification of intraluminal filament technique was reproducible.4. the 2-h MCAO mice developed reproducible neurological deficit and infarcts involving the cerebral cortex and the striatum. The 30 min MCAO mice developed selective neuronal injury in the striatum, and none of them showed residual neurological deficit.5. The injection of TAT-Ngb significantly decreased infarct volume and improved neurologic outcomes after 2 h of focal ischemia, it also markedly attenuated the ischemia-induced neuronal injury and apoptosis after 30 min of focal ischemia.These demonstrated that intravenous delivery of TAT-Ngb can protect the brain from mild or moderate ischemic injury.