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

Abstract

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.

Keywords: MRI; behavior analysis; fecal matter transfer; gait analysis; microbiome gut-brain axis; porcine (pig) model; traumatic brain injury.

FIGURE 1

FMT significantly reduced lesion volume and MLS following TBI. Coronal T2W MRI scans revealed minimal to no lesion present in Sham animals at 1 and 7 days (A,B). Hyperintense and hypointense ipsilateral lesioning characteristic of heterogenous TBI were present at 1 and 7 days in TBI non-treated (C,D) and to a lesser degree in FMT treated (E,F) animals. FMT treatment led to a significantly reduced lesion volume (G) and lesioned percent of the ipsilateral hemisphere (H) at 7 days as compared to 1 day post TBI. TBI non-treated animals experienced a significant change in MLS as measured at the falx cerebri (I) and at the septum pellucidum (J), while FMT treated animals showed no changes in MLS. All data is expressed as mean ± SEM. Bar denotes a time effect within treatment. **p < 0.01 and *p < 0.05.

FIGURE 2

FMT significantly reduced hemorrhage volume in TBI piglets. Coronal SWAN images showed hypointense regions indicative of intracerebral blood and blood products (A–F). Sham animals show minimal hemorrhage at 1 day (A) and 7 days (B). TBI non-treated animals showed a slight decrease in hypointense hemorrhagic lesion volume from 1 day (C) to 7 days (D) post TBI whereas FMT treated animals showed a significant reduction in hypointense hemorrhagic lesion volume from 1 day (E) to 7 days (F) post TBI (G). Data is presented as mean ± SEM. **p < 0.01.

FIGURE 3

FMT ameliorates cytotoxic and vasogenic edema. Axial ADC maps showed minimal edema at 1 day (A) and 7 days (B) in Sham animals. However, the percentage of the ipsilateral hemisphere occupied by cytotoxic edema (hypointense regions) was significantly elevated in TBI non-treated (C) and FMT treated (E) animals at 1 day post TBI as compared to Sham (A,G). However, the percentage of the ipsilateral hemisphere occupied by cytotoxic edema was not significantly different between Sham (B) and FMT (F) treated animals at 7 days post TBI, while cytotoxic edema remained significantly elevated in TBI non-treated animals (D,G). An overall treatment effect was reported in the percent of the ipsilateral hemisphere occupied by vasogenic edema (hyperintense regions) in which TBI non-treated animals (C,D) displayed a significantly greater area of hyperintensity whereas there was no difference in vasogenic edema in FMT treated animals (E,F) compared to Sham animals (A,B,H). Data is presented as mean ± SEM. Treatment effect within day is compared to Sham. Bracket indicates treatment difference compared to Sham. **p < 0.01.

FIGURE 4

FMT increases neuron survival in TBI animals at 7 days post TBI. Histological evaluation of NeuN+ neurons in Sham (A), TBI non-treated (B), and FMT treated animals (C) showed TBI non-treated animals demonstrated a significant decrease in NeuN+ neurons relative to Sham animals, while FMT treated animals (C) showed no difference from Sham (D). Histological assessment of Olig2+ oligodendrocytes in Sham (E), TBI non-treated (F), and FMT treated animals (G) showed TBI non-treated animals demonstrated a significant decrease in Olig2+ relative to Sham animals, while FMT treated animals did not (H). Iba1+ activated microglia in Sham (I), TBI non-treated (J), and FMT treated (K) animals showed TBI non-treated and FMT treated animals displayed a significant increase in Iba1+ microglia cells as compared to Sham animals (L). GFAP+ astrocytes in Sham (M), TBI non-treated (N), and FMT treated (O) animals demonstrated TBI non-treated and FMT treated animals showed a significant increase in GFAP+ astrocyte activation as compared to Sham animals (P). DCX+ neuroblasts in Sham (Q), TBI non-treated (R), and FMT treated (S) animals demonstrated TBI non-treated animals showed a significant increase in DCX+ cells as compared to FMT treated and Sham animals (T). Additionally, FMT treated animals had a significant increase in DCX+ cells as compared to Sham (T). All images were captured at 10 × magnification. Data is presented as mean ± SEM. Treatment effects are as compared to Sham. **p < 0.01 and * p < 0.05.

FIGURE 5

FMT prevented pathogenic bacterial increase in feces of FMT treated piglets at acute timepoints. Fecal microbiome analysis of Actinobacillus indolicus (A), Actinomyces howellii (B), Bifidobacterium animalis (C), and Streptococcus hyointestinalis (D) showed that TBI non-treated animals had an acute increase in potentially pathogenic bacteria compared to FMT treated and Sham animals. Data is presented as mean ± SEM. **p < 0.01 and *p < 0.05.

FIGURE 6

FMT increased probiotic cecal bacteria at 7 days post TBI. Cecal microbiome analysis revealed significant increases in probiotic species L. amylovorus (A) and L. mucosae (B) in the cecum of FMT treated animals versus TBI non-treated animals. L. coleohominis (C) and L. pontis (D) were increased in FMT treated animals compared to TBI non-treated and Sham animals. Data is presented as mean ± SEM. **p < 0.01 and *p < 0.05.

FIGURE 7

FMT treatment restores gut villi morphology. At 7 days post TBI, ileum tissue was acquired, and morphological measurements including villi height (red line), (A) and crypt depth (black line), (A) were obtained on 15 intact villi. TBI non-treated animals showed a significant reduction in villi length to crypt depth ratio, while FMT treated animals showed no significant difference (B). No differences were noted between any treatment group in villi length (C) and crypt depth (D). Data is presented as mean ± SEM. Treatment effects are as compared to Sham. **p < 0.01.

FIGURE 8

FMT improves step and stride length following TBI. Step length is the measured distance between heel strikes of opposite hooves (A). At 1 day post TBI a decrease in left front step length was noted in TBI non-treated and FMT treated animals as compared to Sham (B). At 3 days post TBI, TBI non-treated animals displayed a decreased step length in the left front (B) limbs as compared to Sham. At 7 days post TBI, TBI non-treated animals displayed a decreased step length in the left front (B) limbs as compared to Sham and FMT treated animals. However, there was no difference in right front (C), right hind (E) or left hind (D) step length between treatments. Stride length measures the distance between consecutive heel strikes of the same limb (F). At 1 day post TBI, TBI non-treated pigs exhibited a reduced stride length in the right front (H) limb as compared to Sham. At 3 days post TBI, TBI non-treated pigs exhibited a reduced stride length in the left front (G) and right front (H) limb as compared to Sham. At 7 days post TBI, as compared to Sham and FMT treated animals, TBI non-treated animals showed a decreased stride length in the left front (G) and right front (H) limbs. At 7 days post TBI, TBI non-treated animals showed a decreased stride length in the left hind (I) limbs as compared to FMT treated animals. However, there was no difference in right hind stride length between treatments (J). All data is presented as mean ± SEM. Treatment effects are as compared to Sham. *p < 0.05.

FIGURE 9

FMT improved voluntary motor activity and exploratory sniffing behavior following TBI. Representative tracking images of distance traveled (A–C); (red lines), and time sniffing (D–F); (red lines) are shown at 1 day post TBI in Sham (A,D), respectively, TBI non treated (B,E), respectively, and FMT treated (C,F), respectively animals. At 1 and 3 days post TBI, TBI non-treated animals traveled significantly less distance (G), moved at a slower velocity (H), spent less time moving during their trial (I), spent a smaller percent of the trial moving (K), and spent more time not moving (L) as compared to Sham animals, while FMT treated animals were more comparable to sham. Additionally, at 1 day post TBI, TBI non-treated animals spent less time sniffing (J) compared to Sham. Data is presented as mean ± SEM. Treatment effects are as compared to Sham. **p < 0.01 and *p < 0.05.