Beneficial Effects of Human Schwann Cell-Derived Exosomes in Mitigating Secondary Damage After Penetrating Ballistic-Like Brain Injury

Abstract

There is a growing body of evidence that the delivery of cell-derived exosomes normally involved in intracellular communication can reduce secondary injury mechanisms after brain and spinal cord injury and improve outcomes. Exosomes are nanometer-sized vesicles that are released by Schwann cells and may have neuroprotective effects by reducing post-traumatic inflammatory processes as well as promoting tissue healing and functional recovery. The purpose of this study was to evaluate the beneficial effects of human Schwann-cell exosomes (hSC-Exos) in a severe model of penetrating ballistic-like brain injury (PBBI) in rats and investigate effects on multiple outcomes. Human Schwann cell processing protocols followed Current Good Manufacturing Practices (cGMP) with exosome extraction and purification steps approved by the Food and Drug Administration for an expanded access single ALS patient Investigational New Drug. Anesthetized male Sprague-Dawley rats (280-350g) underwent PBBI surgery or Sham procedures and, starting 30 min after injury, received either a dose of hSC-Exos or phosphate-buffered saline through the jugular vein. At 48h after PBBI, flow cytometry analysis of cortical tissue revealed that hSC-Exos administration reduced the number of activated microglia and levels of caspase-1, a marker of inflammasome activation. Neuropathological analysis at 21 days showed that hSC-Exos treatment after PBBI significantly reduced overall contusion volume and decreased the frequency of Iba-1 positive activated and amoeboid microglia by immunocytochemical analysis. This study revealed that the systemic administration of hSC-Exos is neuroprotective in a model of severe TBI and reduces secondary inflammatory injury mechanisms and histopathological damage. The administration of hSC-Exos represents a clinically relevant cell-based therapy to limit the detrimental effects of neurotrauma or other progressive neurological injuries by impacting multiple pathophysiological events and promoting neurological recovery.

Keywords: exosomes; human Schwann cells; inflammasome; inflammation; penetrating ballistic-like brain injury; traumatic brain injury.

FIG. 1.

Nanosight analysis of hSC-derived exosomes. (A) Nanosight nanoparticle analysis of hSC-derived exosomes. (B) Graph demonstrating hSC-derived exosome size. (C) Graph representing the hSC-derived exosome concentration.

FIG. 2.

Transmission electron microcopy (TEM) of hSC-derived exosomes. hSCs were isolated and imaged using TEM.

FIG. 3.

Lesion volume analysis 21 days after penetrating ballistic-like brain injury (PBBI) and hSC-Exos treatment. (A) Representative images showing that hSC-Exos treatment significantly decreases lesion volume 21 days after PBBI between bregma levels -1.08 -1.68. (B) Quantification of lesion area analysis by bregma level using a two-way repeated measures analysis of variance (ANOVA) (n = 10/group). Group comparison was significant (p < 0.05), bregma levels were significant (p < 0.05) but group × bregma was not significant (p = 0.3112). The significance of the group and bregma level changes indicate that overall there are differences between treatment groups as well as changes in the lesion area throughout the neuraxis. (C) PBBI + hSC-Exos treated rats had significantly decreased cavity + perilesional volume compared with PBBI+phosphate-buffered saline (PBS) treated rats at 21 days post-PBBI (n = 10, *p < 0.05) using a Wilcoxon match-pairs signed rank test. (D) One-way ANOVA showed a significant difference in ipsilateral cortical volume between groups (p < 0.0001). Post-hoc analysis showed that PBBI+hSC-Exos treated rats demonstrated a statistically significant increase in ipsilateral lesion volume 21 days post-PBBI compared with PBBI + PBS treated rats (*p < 0.05). Additional group comparisons were significant for PBBI+PBS and Sham+PBS (****p < 0.0001), PBBI+PBS and Sham+hSC-Exos (****p < 0.0001), PBBI+hSC-Exos and Sham+PBS (****p < 0.0001), PBBI+hSC-Exos and Sham+hSC-Exos. (****p < 0.0001). (n = 10/group).

FIG. 4.

Flow cytometry analysis of activated microglia 48h after penetrating ballistic-like brain injury (PBBI) and treatment with hSC-Exos. Representative flow cytometry scatter density plots from cortical tissue of (A) Sham + phosphate-buffered saline (PBS), (B) Sham + hSC-Exos, (C) PBBI + PBS, (D) PBBI + hSC-Exos 48h after PBBI. Quantification of (E) resting microglia, (F) activated microglia, (G) infiltrating CD11b leukocytes. The PBBI + hSC-Exos (PBBI+ hsC-Ex) treatment 48h after PBBI significantly decreased the number of activated microglia (F). There was no significant difference in the number of resting microglia (E) or infiltrating CD11b positive leukocytes (G). Statistical significance was determined using a one-way analysis of variance, n = 5–6/group, *p < 0.05, ns = no significance.

FIG. 5.

Flow cytometry analysis of caspase-1 activation 48h after penetrating ballistic-like brain injury (PBBI) and treatment with hSC-Exos. Representative flow cytometry scatter density plots of caspase-1 activity (FAM-FLICA) versus LIVE/DEAD from cortical tissue of (A) Sham + phosphate-buffered saline (PBS), (B) Sham + hSC-Exos, (C) PBBI + PBS, (D) PBBI + hSC-Exos 48h after PBBI. CD11b positive cells were gated based on high FLICA expression and high LIVE/DEAD expression for pyroptotic cells. Caspase-1 activity in live cells was determined by high FLICA expression and low LIVE/DEAD expression. Quantification of (E) caspase-1 activity live cells and (F) pyropotic cells. There was no significant difference in the number of caspase-1 activity in live cells or cells undergoing pyroptosis. Statistical significance was determined using a one-way analysis of variance, n = 5–6, ns = no significance.

FIG. 6.

Flow cytometry analysis of caspase-1 activation in activated microglia and infiltrating leukocytes 48h after penetrating ballistic-like brain injury (PBBI) and treatment with hSC-Exos. Representative flow cytometry scatter density plots of CD11b+ leukocytes, activated microglia, and resting microglia that have caspase-1 activity or are pyroptotic cells of (A) Sham + phosphate-buffered saline (PBS), (B) Sham + hSC-Exos, (C) PBBI + PBS, (D) PBBI + hSC-Exos 48h after PBBI. Cells that were gated for caspase-1 activity in live cells or pyroptotic cells were further gated into the three groups mentioned above. Quantification of (E) resting microglia, (F) activated microglia, (G) infiltrating CD11b leukocytes that are either caspase-1 active or undergoing pyroptosis. The PBBI + hSC-Exos (PBBI+ hsC-Ex) treatment 48h after PBBI significantly decreased the number of activated microglia (F). There was no significant difference in the number of resting microglia (E) or infiltrating CD11b positive leukocytes (G). Statistical significance was determined using a one-way analysis of variance, n = 5-6, *p < 0.05, ns = no significance.

FIG. 7.

hSC-Exos treatment reduces the number of Iba-1 positive activated and amoeboid microglia 21 days after penetrating ballistic-like brain injury (PBBI). (A) Representative images (60x) of microglia that were used for unbiased stereological counts based on morphology. Cells were classified into four major types: resting, primed, activated, and amoeboid. The scale bars represent 10 μm. (B) Representative lower magnification images (10x) in the injured cortex where higher magnification images were taken. Black arrows point to the perilesion where images were taken. (C) Quantification of Iba-1+ microglia in the ipsilateral cortex. There was an increase in activated and amoeboid cells after PBBI compared with sham-operated groups. There was a significant (p < 0.05) increase in resting and primed microglia and a significant (p < 0.05) reduction in amoeboid in the PBBI group administrated with hSC-Exos. Data are presented as mean ± standard error of the mean. Statistical significance was determined with one-way analysis of variance followed by the Tukey multiple comparison test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. n = 5–6 per group.

FIG. 8.

Immunohistochemical representation of Iba-1 positive microglial morphology changes after penetrating ballistic-like brain injury (PBBI) and hSC-Exos treatment. (A) Sham + phosphate-buffered saline (PBS) and (B) Sham + hSC-Exos treatment rats have increased presence of resting microglia compared with injured groups (Iba1-red). White arrows indicate resting state microglia. (C) PBBI + PBS treated rats have increased activated microglia as demonstrated by white arrow. (D) PBBI + hSC-Exos treatment rats have less activated microglia compared with the PBBI+PBS group as well as increased primed microglia (white arrow) (Iba1-red) in A–D images taken at 60x; scale bar = 16 μm. (E,F) Representative 10x images of perilesional area of injured cortex where higher magnification images were taken (white box); scale bar = 282.27 μm.