Intranasal wnt3a Attenuates Neuronal Apoptosis through Frz1/PIWIL1a/FOXM1 Pathway in MCAO Rats

Abstract

After ischemic stroke, apoptosis of neurons is a primary factor in determining outcome. Wnt3a is a naturally occurring protein that has been shown to have protective effects in the brain for traumatic brain injury. Although wnt3a has been investigated in the phenomena of neurogenesis, anti-apoptosis, and anti-inflammation, it has never been investigated as a therapy for stroke. We hypothesized that the potential neuroprotective agent wnt3a would reduce infarction and improve behavior following ischemic stroke by attenuating neuronal apoptosis and promoting cell survival through the Frizzled-1/PIWI1a/FOXM1 pathway in middle cerebral artery occlusion (MCAO) rats. A total of 229 Sprague Dawley rats were assigned to male, female, and 9-month-old male MCAO or sham groups followed by reperfusion 2 h after MCAO. Animals assigned to MCAO were either given wnt3a or its control. To explore the downstream signaling of wnt3a, the following interventions were given: Frizzled-1 siRNA, PIWI1a siRNA, and PIWI1a-clustered regularly interspaced short palindromic repeats, along with the appropriate controls. Post-MCAO assessments included neurobehavioral tests, infarct volume, Western blot, and immunohistochemistry. Endogenous levels of wnt3a and Frizzled-1/PIWI1a/FOXM1 were lowered after MCAO. The administration of intranasal wnt3a, 1 h after MCAO, increased PIWIL1a and FOXM1 expression through Frizzled-1, reducing brain infarction and neurological deficits at 24 and 72 h. Frizzled-1 and PIWI1a siRNAs reversed the protective effects of wnt3a after MCAO. Restoration of PIWI1a after knockdown of Frizzled-1 increased FOXM1 survival protein and reduced cleaved caspase-3 levels. In summary, wnt3a decreases neuronal apoptosis and improves neurological deficits through Frizzled-1/PIWI1a/FOXM1 pathway after MCAO in rats. Therefore, wnt3a is a novel intranasal approach to decrease apoptosis after stroke. Only 5% of patients receive recombinant tissue plasminogen activator after stroke, and few qualify for mechanical thrombectomy. No neuroprotective agents have been successfully translated to promote neuronal survival in stroke. Thus, using a clinically relevant rat model of stroke, middle cerebral artery occlusion, we explored a novel intranasal administration of wnt3a. wnt3a naturally occurs in the body and crosses the blood-brain barrier, supporting the clinically translatable approach of intranasal administration. Significant neuronal apoptosis occurs during stroke, and wnt3a shows promise due to its antiapoptotic effects. We investigated whether wnt3a mediates its poststroke effects via Frizzled-1 and the impact on its downstream signaling molecules, PIWI1a and FOXM1, in apoptosis. Elucidating the mechanism of wnt3a will identify additional pharmacological targets and further understanding of stroke.

Ezetimibe Attenuates Oxidative Stress and Neuroinflammation via the AMPK/Nrf2/TXNIP Pathway after MCAO in Rats

“…. The transient MCAO model was used in male Sprague-Dawley rats as previously described [28]. Briefly, anesthesia was induced intraperitoneally with a mixture of ketamine (80 mg/kg, K2573; Sigma-Aldrich, St. Louis, MO, USA) and xylazine (10 mg/kg, X1126; Sigma-Aldrich, St. Louis, MO, USA).…”

“…Intracerebroventricular (i.c.v.) administration was performed as previously described [28]. Briefly, rats were placed in a stereotaxic apparatus under 2.5% isoflurane anesthesia.…”

IRE1α inhibition attenuates neuronal pyroptosis via miR-125/NLRP1 pathway in a neonatal hypoxic-ischemic encephalopathy rat model

“…The NLRP1 CRISPR activation plasmid (0.4 μg/pup, NLRP1 SAM guide RNA, Qiagen, Quote#U8376ED300) or control CRISPR activation plasmid (0.4 μg/pup, NLRP1 SAM guide RNA negative control, Qiagen, Quote#U8376ED300) was given via intracerebroventricular injection as described above at 24 h before HI. The time point for miRNA inhibitor and CRISPR injection was selected based on previous literature [32,33]. According to the manufacturers' protocol, the phenotypic effects of the products are normally assessed 24-72 h after delivery.…”

NT-4 attenuates neuroinflammation via TrkB/PI3K/FoxO1 pathway after germinal matrix hemorrhage in neonatal rats

“…FoxO1 CRISPR (Santa Cruz Biotechnology) was suspended in the plasmid transfection medium and activated with transfection reagent, totaling 2 μg per animal. The control scramble CRISPR was prepared following the same steps and a total of 2 μg per animal was given intracerebroventricularly [30,35]. Animals were randomly divided into the following groups: sham group (n = 6), GMH + vehicle group (n = 6, i.n.…”