Fecal microbial transplantation limits neural injury severity and functional deficits in a pediatric piglet traumatic brain injury model.

Pediatric traumatic brain injury (TBI) is a leading cause of death and disability in children. Due to bidirectional communication between the brain and gut microbial population, introduction of key gut bacteria may mitigate critical TBI-induced secondary injury cascades, thus lessening neural damage and improving functional outcomes. The objective of this study was to determine the efficacy of a daily fecal microbial transplant (FMT) to alleviate neural injury severity, prevent gut dysbiosis, and improve functional recovery post TBI in a translational pediatric piglet model. Male piglets at 4-weeks of age were randomly assigned to Sham + saline, TBI + saline, or TBI + FMT treatment groups. A moderate/severe TBI was induced by controlled cortical impact and Sham pigs underwent craniectomy surgery only. FMT or saline were administered by oral gavage daily for 7 days. MRI was performed 1 day (1D) and 7 days (7D) post TBI. Fecal and cecal samples were collected for 16S rRNA gene sequencing. Ipsilateral brain and ileum tissue samples were collected for histological assessment. Gait and behavior testing were conducted at multiple timepoints. MRI showed that FMT treated animals demonstrated decreased lesion volume and hemorrhage volume at 7D post TBI as compared to 1D post TBI. Histological analysis revealed improved neuron and oligodendrocyte survival and restored ileum tissue morphology at 7D post TBI in FMT treated animals. Microbiome analysis indicated decreased dysbiosis in FMT treated animals with an increase in multiple probiotic Lactobacilli species, associated with anti-inflammatory therapeutic effects, in the cecum of the FMT treated animals, while non-treated TBI animals showed an increase in pathogenic bacteria, associated with inflammation and disease such in feces. FMT mediated enhanced cellular and tissue recovery resulted in improved motor function including stride and step length and voluntary motor activity in FMT treated animals. Here we report for the first time in a highly translatable pediatric piglet TBI model, the potential of FMT treatment to significantly limit cellular and tissue damage leading to improved functional outcomes following a TBI.

Cooperative activation of PDK1 and AKT by MAPK4 enhances cancer growth and resistance to therapy.

Phosphoinositide-dependent kinase-1 (PDK1) is a master kinase of the protein A, G, and C (AGC) family kinases that play important roles in regulating cancer cell proliferation, survival, and metabolism. Besides phosphorylating/activating AKT at the cell membrane in a PI3K-dependent manner, PDK1 also exhibits constitutive activity on many other AGC kinases for tumor-promoting activity. In the latter case, PDK1 protein levels dominate its activity. We previously reported that MAPK4, an atypical MAPK, can PI3K-independently promote AKT activation and tumor growth. Here, using triple-negative breast cancer (TNBC) cell models, we demonstrate that MAPK4 can also enhance PDK1 protein synthesis, thus phosphorylate/activate PDK1 substrates beyond AKT. This new MAPK4-PDK1 axis alone lacks vigorous tumor-promoting activity but cooperates with our previously reported MAPK4-AKT axis to promote tumor growth. Besides enhancing resistance to PI3K blockade, MAPK4 also promotes cancer cell resistance to the more stringent PI3K and PDK1 co-blockade, a recently proposed therapeutic strategy. Currently, there is no MAPK4 inhibitor to treat MAPK4-high cancers. Based on the concerted action of MAPK4-AKT and MAPK4-PDK1 axis in promoting cancer, we predict and confirm that co-targeting AKT and PDK1 effectively represses MAPK4-induced cancer cell growth, suggesting a potential therapeutic strategy to treat MAPK4-high cancers.

Tanshinone IIA-loaded nanoparticles and neural stem cell combination therapy improves gut homeostasis and recovery in a pig ischemic stroke model.

Impaired gut homeostasis is associated with stroke often presenting with leaky gut syndrome and increased gut, brain, and systemic inflammation that further exacerbates brain damage. We previously reported that intracisternal administration of Tanshinone IIA-loaded nanoparticles (Tan IIA-NPs) and transplantation of induced pluripotent stem cell-derived neural stem cells (iNSCs) led to enhanced neuroprotective and regenerative activity and improved recovery in a pig stroke model. We hypothesized that Tan IIA-NP + iNSC combination therapy-mediated stroke recovery may also have an impact on gut inflammation and integrity in the stroke pigs. Ischemic stroke was induced, and male Yucatan pigs received PBS + PBS (Control, n = 6) or Tan IIA-NP + iNSC (Treatment, n = 6) treatment. The Tan IIA-NP + iNSC treatment reduced expression of jejunal TNF-α, TNF-α receptor1, and phosphorylated IkBα while increasing the expression of jejunal occludin, claudin1, and ZO-1 at 12 weeks post-treatment (PT). Treated pigs had higher fecal short-chain fatty acid (SCFAs) levels than their counterparts throughout the study period, and fecal SCFAs levels were negatively correlated with jejunal inflammation. Interestingly, fecal SCFAs levels were also negatively correlated with brain lesion volume and midline shift at 12 weeks PT. Collectively, the anti-inflammatory and neuroregenerative treatment resulted in increased SCFAs levels, tight junction protein expression, and decreased inflammation in the gut.

Tanshinone IIA-Loaded Nanoparticle and Neural Stem Cell Therapy Enhances Recovery in a Pig Ischemic Stroke Model.

Induced pluripotent stem cell-derived neural stem cells (iNSCs) are a multimodal stroke therapeutic that possess neuroprotective, regenerative, and cell replacement capabilities post-ischemia. However, long-term engraftment and efficacy of iNSCs is limited by the cytotoxic microenvironment post-stroke. Tanshinone IIA (Tan IIA) is a therapeutic that demonstrates anti-inflammatory and antioxidative effects in rodent ischemic stroke models and stroke patients. Therefore, pretreatment with Tan IIA may create a microenvironment that is more conducive to the long-term survival of iNSCs. In this study, we evaluated the potential of Tan IIA drug-loaded nanoparticles (Tan IIA-NPs) to improve iNSC engraftment and efficacy, thus potentially leading to enhanced cellular, tissue, and functional recovery in a translational pig ischemic stroke model. Twenty-two pigs underwent middle cerebral artery occlusion (MCAO) and were randomly assigned to a PBS + PBS, PBS + iNSC, or Tan IIA-NP + iNSC treatment group. Magnetic resonance imaging (MRI), modified Rankin Scale neurological evaluation, and immunohistochemistry were performed over a 12-week study period. Immunohistochemistry indicated pretreatment with Tan IIA-NPs increased iNSC survivability. Furthermore, Tan IIA-NPs increased iNSC neuronal differentiation and decreased iNSC reactive astrocyte differentiation. Tan IIA-NP + iNSC treatment enhanced endogenous neuroprotective and regenerative activities by decreasing the intracerebral cellular immune response, preserving endogenous neurons, and increasing neuroblast formation. MRI assessments revealed Tan IIA-NP + iNSC treatment reduced lesion volumes and midline shift. Tissue preservation and recovery corresponded with significant improvements in neurological recovery. This study demonstrated pretreatment with Tan IIA-NPs increased iNSC engraftment, enhanced cellular and tissue recovery, and improved neurological function in a translational pig stroke model.

Changes in Oral Microbial Diversity in a Piglet Model of Traumatic Brain Injury.

Dynamic changes in the oral microbiome have gained attention due to their potential diagnostic role in neurological diseases such as Alzheimer's disease and Parkinson's disease. Traumatic brain injury (TBI) is a leading cause of death and disability in the United States, but no studies have examined the changes in oral microbiome during the acute stage of TBI using a clinically translational pig model. Crossbred piglets (4-5 weeks old, male) underwent either a controlled cortical impact (TBI, n = 6) or sham surgery (sham, n = 6). The oral microbiome parameters were quantified from the upper and lower gingiva, both buccal mucosa, and floor of the mouth pre-surgery and 1, 3, and 7 days post-surgery (PS) using the 16S rRNA gene. Faith's phylogenetic diversity was significantly lower in the TBI piglets at 7 days PS compared to those of sham, and beta diversity at 1, 3, and 7 days PS was significantly different between TBI and sham piglets. However, no significant changes in the taxonomic composition of the oral microbiome were observed following TBI compared to sham. Further studies are needed to investigate the potential diagnostic role of the oral microbiome during the chronic stage of TBI with a larger number of subjects.

Intracisternal administration of tanshinone IIA-loaded nanoparticles leads to reduced tissue injury and functional deficits in a porcine model of ischemic stroke.

BACKGROUND: The absolute number of new stroke patients is annually increasing and there still remains only a few Food and Drug Administration (FDA) approved treatments with significant limitations available to patients. Tanshinone IIA (Tan IIA) is a promising potential therapeutic for ischemic stroke that has shown success in pre-clinical rodent studies but lead to inconsistent efficacy results in human patients. The physical properties of Tan-IIA, including short half-life and low solubility, suggests that Poly (lactic-co-glycolic acid) (PLGA) nanoparticle-assisted delivery may lead to improve bioavailability and therapeutic efficacy. The objective of this study was to develop Tan IIA-loaded nanoparticles (Tan IIA-NPs) and to evaluate their therapeutic effects on cerebral pathological changes and consequent motor function deficits in a pig ischemic stroke model. RESULTS: Tan IIA-NP treated neural stem cells showed a reduction in SOD activity in in vitro assays demonstrating antioxidative effects. Ischemic stroke pigs treated with Tan IIA-NPs showed reduced hemispheric swelling when compared to vehicle only treated pigs (7.85 ± 1.41 vs. 16.83 ± 0.62%), consequent midline shift (MLS) (1.72 ± 0.07 vs. 2.91 ± 0.36 mm), and ischemic lesion volumes (9.54 ± 5.06 vs. 12.01 ± 0.17 cm3) when compared to vehicle-only treated pigs. Treatment also lead to lower reductions in diffusivity (-37.30 ± 3.67 vs. -46.33 ± 0.73%) and white matter integrity (-19.66 ± 5.58 vs. -30.11 ± 1.19%) as well as reduced hemorrhage (0.85 ± 0.15 vs 2.91 ± 0.84 cm3) 24 h post-ischemic stroke. In addition, Tan IIA-NPs led to a reduced percentage of circulating band neutrophils at 12 (7.75 ± 1.93 vs. 14.00 ± 1.73%) and 24 (4.25 ± 0.48 vs 5.75 ± 0.85%) hours post-stroke suggesting a mitigated inflammatory response. Moreover, spatiotemporal gait deficits including cadence, cycle time, step time, swing percent of cycle, stride length, and changes in relative mean pressure were less severe post-stroke in Tan IIA-NP treated pigs relative to control pigs. CONCLUSION: The findings of this proof of concept study strongly suggest that administration of Tan IIA-NPs in the acute phase post-stroke mitigates neural injury likely through limiting free radical formation, thus leading to less severe gait deficits in a translational pig ischemic stroke model. With stroke as one of the leading causes of functional disability in the United States, and gait deficits being a major component, these promising results suggest that acute Tan IIA-NP administration may improve functional outcomes and the quality of life of many future stroke patients.

Dynamic Changes in the Gut Microbiome at the Acute Stage of Ischemic Stroke in a Pig Model.

Stroke is a major cause of death and long-term disability affecting seven million adults in the United States each year. Recently, it has been demonstrated that neurological diseases, associated pathology, and susceptibility changes correlated with changes in the gut microbiota. However, changes in the microbial community in stroke has not been well characterized. The acute stage of stroke is a critical period for assessing injury severity, therapeutic intervention, and clinical prognosis. We investigated the changes in the gut microbiota composition and diversity using a middle cerebral artery (MCA) occlusion ischemic stroke pig model. Ischemic stroke was induced by cauterization of the MCA in pigs. Blood samples were collected prestroke and 4 h, 12 h, 1 day, and 5 days poststroke to evaluate circulating proinflammatory cytokines. Fecal samples were collected prestroke and 1, 3, and 5 days poststroke to assess gut microbiome changes. Results showed elevated systemic inflammation with increased plasma levels of tumor necrosis factor alpha at 4 h and interleukin-6 at 12 h poststroke, relative to prestroke. Microbial diversity and evenness were reduced at 1 day poststroke compared to prestroke. Microbial diversity at 3 days poststroke was negatively correlated with lesion volume. Moreover, beta-diversity analysis revealed trending overall differences over time, with the most significant changes in microbial patterns observed between prestroke and 3 days poststroke. Abundance of the Proteobacteria was significantly increased, while Firmicutes decreased at 3 days poststroke, compared to prestroke populations. Abundance of the lactic acid bacteria Lactobacillus was reduced at 3 days poststroke. By day 5, the microbial pattern returned to similar values as prestroke, suggesting the plasticity of gut microbiome in an acute period of stroke in a pig model. These findings provide a basis for characterizing gut microbial changes during the acute stage of stroke, which can be used to assess stroke pathology and the potential development of therapeutic targets.

Perinatal Docosahexaenoic Acid Supplementation Improves Cognition and Alters Brain Functional Organization in Piglets.

Epidemiologic studies associate maternal docosahexaenoic acid (DHA)/DHA-containing seafood intake with enhanced cognitive development; although, it should be noted that interventional trials show inconsistent findings. We examined perinatal DHA supplementation on cognitive performance, brain anatomical and functional organization, and the brain monoamine neurotransmitter status of offspring using a piglet model. Sows were fed a control (CON) or a diet containing DHA (DHA) from late gestation throughout lactation. Piglets underwent an open field test (OFT), an object recognition test (ORT), and magnetic resonance imaging (MRI) to acquire anatomical, diffusion tensor imaging (DTI), and resting-state functional MRI (rs-fMRI) at weaning. Piglets from DHA-fed sows spent 95% more time sniffing the walls than CON in OFT and exhibited an elevated interest in the novel object in ORT, while CON piglets demonstrated no preference. Maternal DHA supplementation increased fiber length and tended to increase fractional anisotropy in the hippocampus of offspring than CON. DHA piglets exhibited increased functional connectivity in the cerebellar, visual, and default mode network and decreased activity in executive control and sensorimotor network compared to CON. The brain monoamine neurotransmitter levels did not differ in healthy offspring. Perinatal DHA supplementation may increase exploratory behaviors, improve recognition memory, enhance fiber tract integrity, and alter brain functional organization in offspring at weaning.

Characterization of tissue and functional deficits in a clinically translational pig model of acute ischemic stroke.

The acute stroke phase is a critical time frame used to evaluate stroke severity, therapeutic options, and prognosis while also serving as a major tool for the development of diagnostics. To further understand stroke pathophysiology and to enhance the development of treatments, our group developed a translational pig ischemic stroke model. In this study, the evolution of acute ischemic tissue damage, immune responses, and functional deficits were further characterized. Stroke was induced by middle cerebral artery occlusion in Landrace pigs. At 24 h post-stroke, magnetic resonance imaging revealed a decrease in ipsilateral diffusivity, an increase in hemispheric swelling resulting in notable midline shift, and intracerebral hemorrhage. Stroke negatively impacted white matter integrity with decreased fractional anisotropy values in the internal capsule. Like patients, pigs showed a reduction in circulating lymphocytes and a surge in neutrophils and band cells. Functional responses corresponded with structural changes through reductions in open field exploration and impairments in spatiotemporal gait parameters. Characterization of acute ischemic stroke in pigs provided important insights into tissue and functional-level assessments that could be used to identify potential biomarkers and improve preclinical testing of novel therapeutics.

Depletion of regulator-of-G-protein signaling-10 in mice exaggerates high-fat diet-induced insulin resistance and inflammation, and this effect is mitigated by dietary green tea extract.

The interaction between insulin resistance and inflammation plays a central role in the development of chronic diseases, although the mechanism is not fully understood. We previously demonstrated that regulator of G-protein signaling-10 (RGS10) protein is a negative modulator of the inflammatory response in macrophages and microglia. Because inflammation is a critical component in the development of high fat diet-induced insulin resistance, in this study we investigated whether RGS10 is involved in the diet-dependent regulation of glucose tolerance and insulin sensitivity. We hypothesized that the absence of RGS10 would exaggerate high-fat diet (HFD)-induced insulin resistance and inflammation response. Our results showed that RGS10 knockout (KO) mice fed a HFD gained significantly more weight and developed severe insulin resistance compared to wild-type (WT) mice fed HFD. Furthermore, compared to WT HFD-fed mice, KO mice fed the HFD displayed inflammatory phenotypes such as decreased adipose tissue expression of the anti-inflammatory M2 markers YM1 and Fizz1 and increased expression of the proinflammatory M1 cytokine interleukin 6 in adipose and CD11b, CD68 and interleukin 1ß in liver tissues. The impact of RGS10 deficiency on the exaggeration of HFD-induced insulin resistance and inflammation was ameliorated by oral consumption of green tea extract. Our results demonstrate that RGS10 is an important part of a protective mechanism involved in in regulating metabolic homeostasis by reducing inflammatory responses, which could potentially lead to an innovative new approach targeting inflammation and insulin resistance.