Acute social defeat-induced neuroinflammation in the vmPFC of Syrian hamsters via microglial activation

Authors: J. Alex Grizzell, Thomas T. Clarity, Matthew S. Jenkins, Matthew A. Cooper
​Affiliation: Department of Psychology and NeuroNET Research Center, The University of Tennessee, Knoxville

TAKE HOME FINDINGS:
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Consequences of acute social stress may be mediated by immune system within the brain. 

Microglia in prefrontal cortex are activated by acute trauma alone and "primed" for greater future activation. 

Acute social stress causes cellular degeneration within prefrontal cortex.
​

Introduction

  • Psychological stress and neuroinflammation bidirectionally influence one another - thereby implicating neuroinflammatory pathways as contributing factors to the etiologies of various stress-related psychopathologies.
  • Exposure to chronic stress results in elevated markers of microglial activity, as measured by ionized calcium binding adaptor protein-1 (Iba-1) expression.
  • Microglia, which can be halted by minocycline administration, contribute to tissue degradation via proinflammation, oxidative stress, and phagocytosis.
  • Little research probes the effects of acute stress on central immune activity.
  • Acute tailshock can prime microglia to exhibit an enhanced proinflammatory response to a subsequent immune challenge (i.e. Lipopolysaccharide) (1).
  • The prefrontal cortex (PFC) is a critical brain region for stress coping. Chronic stress-induced inflammation alters neuronal morphology in the medial PFC (2).

Methods

Animals: Adult, male Syrian hamsters (Mesocricetus auratus​)
  1. Acute Social Defeat:  Subjects from each experiment were exposed to three, 5-minute aggressive encounters in the home cages of three, separate, larger, sexually experienced, hamsters that were prescreened for aggression. Encounters occurred at 5-minute intervals. Control hamsters were placed in empty aggressors' cages during this time to mirror the defeat procedure yet control for olfactory cues and novel cage exposure. 
  2. LPS Injection: In Experiment 1, 24-hours following social defeat, the effects of stress-induced priming of microglia were assessed by exposure to an immune challenge via intraperitoneal injection of the endotoxin lipopolysaccharide (LPS) at 0, 20, 100, and 500 ug/kg doses. Animals were monitored during this time to determine degree of 'sickness behavior' before euthanasia 4-hours later. 
  3. Minocycline Treatment: In Experiment 3, hamsters were given either water or 4mg/ml of the microglial inhibitor, minocycline, in their drinking water (to average an approximate dose of 130mg/kg/day). Treatment started 48-hours prior to acute social defeat stress and persisted throughout the experiment until euthanasia 48-hours after defeat. Minocycline was mixed within autoclaved tap water with pH adjusted to 7.4. Consumption rates were taken twice daily and solution was changed daily. Minocycline solutions were administered in drip-resistant water bottles that were covered due to light sensitivity.
  4. Social Avoidance Behavioral Assays: In Experiment 3, hamsters were exposed to a social interaction test (SIT) and conditioned defeat (CD) test in a counterbalanced manner 24 hours after defeat and with a 30-60 minute interassay interval. SITs comprised of two 5-minute trials wherein the subjects were placed in a novel cage with a perforated box that was empty (Trial 1 or "T1") or contained a novel conspecific (T2) with minimal intertrial intervals. CD tests comprised one 5-minute trial wherein a juvenile conspecific was placed into the home cage of the subject. For each test, the following behaviors were scored to primarily index social approach and submissive/defensive posturing:
    1. ​Flees/Avoids (defensive/submissive behavior)
    2. Stretch Attends (defensive/risk assessment)
    3. Tail up (submissive)
    4. Submissive monitoring
    5. Paws up (defensive behavior)
    6. Time spent investigating conspecific (social approach)
    7. Time spent avoiding conspecific (social avoidance)

      Other behaviors scored include flank-marks (territoriality), self-grooming, and various aggressive behaviors. All behaviors were quantified via digital video recording and through the use of Noldus Observer by trained experimenters blind to treatment conditions. 

  5. Euthanasia: All animals were transcardially perfused with 4% paraformaldehyde solution and brains extracted for immunohistochemical analyses. 
    1. For Experiment 1, this occurred 4-hours after LPS injection, which was 28-hours after social defeat. 
    2. For Experiment 2, this occurred 7-days after social defeat.
    3. For Experiment 3, this occurred 24-hours after SIT and CD tests, which was 48-hours after social defeat. 
  6. Immunolabeling: For Experiments 1-2, microglia were quantified via immunolabeling of the ionized calcium binding adaptor protein-1 (Iba-1) via immunohistochemical techniques in slices containing the ventromedial prefrontal cortex (vmPFC) using a rabbit monoclonal anti-Iba-1 primary antibody (1:10,000; Abcam - ab178846). For Experiment 3, cellular degeneration of the vmPFC was quantified via an Amino Cupric (Silver Stain) using a proprietary approach developed by NeuroScience Associates (NSA; Knoxville, TN). Histology for Experiments 1-2 was conducted by authors JAG and TTC at The University of Tennesse, Knoxville and Experiment 3 at NSA in a training collaboration with JAG. 
  7. Image Capture: For all experiments, only the infra (IL) and prelimbic (PL) subregions of the vmPFC were analyzed. Briefly, with a 20x objective, two images (dorsal and ventral) encompassing all cortical layers of each subregion were captured per hemisphere (i.e. 2 images/subregion; 4 images/hemisphere).  This occurred in across a rostro-caudal axis with approximately 4-6 brain slices/animal (i.e. 6 for Experiments 1-2, 4 for Experiment 3).  
  8. Image Analyses: For Experiments 1-2, microglia were analyzed using Image J (NIH) to determine optical density of Iba-1 expression. Qualitative appraisals of morphometry were conducted by two experimenters trained to >90% reliability based on those depicted below (as adapted from Hinwood et al., 2012). For Experiment 3, signals for degenerated cells (i.e. neurons, astrocytes) were counted if occurring within the image window (see Histology section below). Due to presence of artifact, collective degeneration cannot be quantified using image analysis software. Therefore, qualitative appraisals of degeneration were scored on a 1-4 scale where 1 = little or no silver staining (i.e. little if any degeneration) and 4 = maximal silver staining (i.e. extreme degeneration).

Image adapted from Hinwood et al., 2012. This served as guide for qualitative appraisals of microglia morphology and thus activation state. 

Experiment 1

Representative Histology

20x images of LPS- (or saline-) injected animals that either did not (left panel) or did (right panel) receive acute social defeat stress. Images depict representative Iba-1 expression 4-hours after intraperitoneal injection of LPS (or saline), which occurred 24-hours following stress. Measurement bar in bottom left = 100um.

Results

Following social defeat stress, animals were injected with 0, 20, 100, or 500 ug/kg of LPS and euthanized 4-hours later. Presented here is the optical density of Iba-1 expression in the infralimbic subregion of the vmPFC.
Shown in white = no stress; orange = acute social defeat​
The asterisk denotes a significant main effect of LPS treatment. 
​n=7-10/group 
Presented here is the optical density of Iba-1 expression in the prelimbic subregion of the vmPFC.
The pound sign denotes a significant main effect of social defeat.
Shown in white = no stress; orange = acute social defeat​​
n=7-10/group
Qualitative appraisals of microglial morphology states based on schematics shown in Hinwood et al., 2012 (above) indicate that acute stress induces morphological changes in microglia, as does treatment with LPS.
The asterisk and pound sign denote significant main effects of LPS and social defeat.
Shown in white = no stress; orange = acute social defeat​
​n=7-10/group

Conclusions from Experiment 1:

  1. Acute social defeat appears to increase optical density of Iba-1 and induce morphological changes of microglia toward a proinflammatory state within the vmPFC. 
  2. Acute social defeat appears to "prime" an enhanced microglial response to future immune challenges. This is particularly detectable at the 20ug/kg dosage, as there is likely a ceiling effect at higher doses.  
  3. Future directions include: probing the defeat- and LPS-induced expression of CD68, a marker of phagocytotic activity present in microglia. 

Experiment 2

A) Optical density of Iba-1 expression 7 days following defeat. No significant differences were observed in either the infra- or prelimbic cortices of the vmPFC.
B) Qualitative appraisals of microglial morphology. No significant differences were observed. Data are presented as pooled across vmPFC subregions. 
Shown in white = no stress; orange = acute social defeat stress
​n=10/group

Conclusions from Experiment 2:

​We detected no evidence that acute stress induces prolonged activation of microglia, including a hyper-arrested ramification state as discussed by Hinwood et al., 2012. This was evidenced by no prolonged elevation of Iba-1 expression or changes in morphology 7 days after acute social defeat stress.

Experiment 3

To determine whether microglia cause cellular damage within the vmPFC in a manner that might influence social avoidance behavior following acute social defeat, we treated a subset of hamsters with the antibiotic minocycline which has been shown to halt microglial activity. 

Animals were treated for 48 hours with minocycline in drinking water (as discussed above) and subjected to a social defeat. 24 hours later, animals were tested in a conditioned defeat test and social interaction test in a counterbalanced manner with continuous minocycline treatment and were euthanized 24 hours later for IHC quantification of cellular degeneration using a silver stain.  
Qualitative appraisals of collective degeneration within the vmPFC indicate a main effect of stress with a marginal reduction by minocycline, which halts microglial activity.  This suggests that acute stress induces cellular degeneration which may be reduced with minocycline administration. Degeneration was approximated on a scale of 1-4 wherein 1=minimal degeneration and 4=maximal degeneration. n=8-12/group.   
Picture
Analyses of Amino Cupric (Silver Stain) indicates a greater collection of degenerated cells within the vmPFC of stressed hamsters having been treated with regular drinking water. This effect was protected by minocycline administration, suggesting that stress drives degeneration of what appear to be astrocytes in a microglial-dependent manner.
n=8-12/group