Shipment Stress in Early Life Aggravates Disease Pathogenesis in Mice with Experimental Autoimmune Encephalomyelitis: Support for a Two-Hit Hypothesis of Multiple Sclerosis Etiology

Visual impairments are one of the earliest diagnosed symptoms of multiple sclerosis (MS). The onset and progression of vision loss in MS may be influenced by cumulative psychophysiological stress. Here, we used a two-hit model of stress in female mice to determine if early life stress (ELS) influences the clinical severity of experimental autoimmune encephalomyelitis (EAE) later in life. We hypothesized that ELS caused by animal transportation during early postnatal development represents a co-factor which can exacerbate the disease severity of EAE. Adult EAE mice with ELS displayed more severe clinical signs and delayed recovery compared to non-stressed EAE mice. ELS also diminished visual acuity measured by optokinetic responses, locomotion and exploratory behaviours in EAE mice. Notably, ELS caused earlier onset of visual impairments in EAE. Exacerbated functional impairments in stressed EAE mice were highly correlated with circulating corticosterone levels. The findings show that the progression of induced EAE (second hit) in adulthood can be significantly impacted by adverse early life experiences (first hit). The observations emphasize the importance of comprehensive behavioural testing, including non-motor functions, to enhance the translational value of preclinical animal models of MS. Moreover, shipment stress of laboratory animals should be considered a necessary variable in preclinical MS research. The consideration of cumulative lifetime stresses provides a new perspective of MS pathogenesis within a personalized medicine framework.


Introduction
Multiple sclerosis (MS) is a chronic immune-mediated demyelinating disease which is characterized by progressive neurodegeneration and neurological disability. One of the earliest symptoms of MS is optic neuritis, which often results in significant vision loss (Redler   ) showed that postnatal stress synergistically exacerbates the severity of experimental MS in rats via altering epigenetic regulatory pathways suggesting that stress can represent a significant risk factor for symptomatic deterioration in clinical MS.
Although females are 2-3 times more susceptible to MS than males (Doss ). In the present study, we investigated whether ELS induced by psychophysiological stress in female mice can diminish recovery in a mild form of EAE. We propose that psychophysiological and immune stresses present a two-hit model for symptomatic EAE with the following components: (i) an early-life psychophysiological challenge induced by shipment stress (the first hit), and (ii) an immune insult induced by EAE (the second hit) later in life. We hypothesized that ELS leads to greater clinical severity of EAE in adulthood and HPA-axis hyperactivity in female mice. The findings provide the first evidence that adverse early life experiences can exacerbate the consequences of a neuroimmunological insult later in life, proposing a mechanism for risk susceptibility to MS in adulthood. Figure 1 illustrates CORT levels in all groups across different time points (pre-induction, day 19 and day 27) and spleen weight. Six mice in different groups were excluded from the blood sampling and CORT analysis due to technical issues. A Kruskal-Wallis H test showed no significant differences between groups in pre-induction (baseline) CORT (p=0.394; panel A).

Early Life Stress Reduces Recovery Following EAE
EAE disease progression is shown in Figure 2A. Spearman's rho; Figure 2 panel C) suggesting that greater CDI in the Stress+EAE animals was associated with higher levels of CORT at the chronic time point post-induction (day 27). It appears that EAE in stressed mice followed the characteristic symptomatic progression with slower recovery process (delayed remission) influenced by ELS exposure.  Figure 4 panel C).

Discussion
Evidence is lacking for the cumulative impact of early postnatal adversities and neuroimmunological challenges in adulthood, such as MS. In the present study, we showed that early life shipment stress in female mice leads to aggravated risk and diminished recovery following EAE in later life. Notably, ELS promotes earlier onset of vision impairments, one of the earliest symptoms of MS in patients. Mice with ELS also displayed higher susceptibility to the clinical course and severity of a mild form of EAE compared to non-stressed mice (Fig.5).
The differential effect among groups suggests that even a moderate stress during the early postnatal days (first hit) promotes a vulnerable phenotype that is more susceptible to an immunological challenge (second hit), followed by exacerbated deficits and reduced recovery in both sensory and motor abilities. The heightened susceptibility to EAE in stressed mice was also reflected in their greater level of CORT responses to the disease during the chronic phase. Our findings reveal synergistic interactions between two stressors with cumulative impact on EAE severity, and support theories that onset and progression of pathogenesis in many diseases involve multiple or accumulated psychophysiological hits (Giovanoli  ).
Consequently, the long-lasting reduced frequency threshold in the stressed mice likely represents the greater impact of EAE-induced neuroinflammatory processes on the function of these subcortical pathways and the retinal output. Thus, the present findings enable insights into the dynamic interactions between cytokine function and stress hormone in response to homeostatic challenges.
Importantly, visual acuity during the CW rotation (left-eye movement) in both groups was negatively correlated with CDI. This likely indicates an asymmetry in optic neuritis, gliosis and synaptic impairments rather than systemic inflammation (Joly damage. Thus, the heightened levels of CORT in association with frequency threshold following EAE induction may also represent a neurohormonal response that in turn alleviates some of the harmful consequences of the neuroinflammation. Consistent with the clinical, hormonal and optokinetic findings, the assessment of locomotion also indicated that stressed mice traveled significantly less than non-stressed animals in the chronic phase of EAE (post-induction day 25). Although EAE has the potential to decrease exploratory behaviours in females more than males at the chronic time point (Faraji ).
Despite the frequent use of animal shipment procedures in preclinical studies, the impact of transportation stress is rarely considered in experimental design and data interpretation. ), may exert cumulative and potentially synergistic effects in pathological conditions. The data support the two-hit model of stress that initial exposure to an early postnatal adversity such as shipping stress in rodents can render the offspring more susceptible to the pathological consequences of a second insult such as EAE. Thus, early postnatal shipping stress and EAE in adulthood caused synergistic effects through which stressed mice suffered earlier onset of vision impairments and more severe motor disability when exposed to EAE than non-stressed animals. Notably, motor deficiency in ELS mice became evident only on post-induction days 21 onward ( Fig. 2A) ) that may influence the response to neurological conditions later in life. ELS also disrupts HPA-axis responsiveness in mice, and is associated with increased peripheral inflammatory cytokine secretion and cytokine levels in brain tissue in adulthood (Hohmann  ).
The present findings show that early life shipment may re-program HPA axis activity with lasting consequences for functional loss and recovery in EAE animals. Early life adversities may disrupt the balance between the function and expression of glucocorticoid receptors (GR) in the brain (de ), however, its effectiveness in the EAE model remains to be shown.

Conclusion
This study revealed compounding effects of ELS and EAE in adult female mice. The two-hit hypothesis suggests that exposure to ELS may challenge immune or neural systems so that a subsequent insult later in life invokes a potentiated immunological and/or neurological response. It is likely that two hits by stress interact in a synergistic manner to increase the risk or severity of a pathological condition. Here we showed that shipment of laboratory mice in early life re-programs HPA axis function in a way to contribute to earlier and more severe functional

Animals
The experimental design is illustrated in Figure 1

Early Life Shipment Stress
Mice in stress groups (n=17) were shipped from an external breeder (Charles River,

Saint-Constant, QC, Canada) to the Canadian Centre for Behavioural Neuroscience (CCBN) in
Lethbridge, Alberta at P5-10 as a multidimensional displacement stressor ). Briefly, shipped animals were placed into paper crates either the shipping day or the day before, depending upon the time of airplane departure. They were then transported by truck to the airport in a temperature-controlled space. Crates were loaded into the airplane cargo compartment which maintained the ambient climate similar to the passenger cabin (temperature 20°C, pressure 8,000 ft ASL). Flight duration varied from 7 to 9 hours. Upon landing, crates were transported by a passenger car to the destination. It was shown that early life shipment represents a multidimensional stressor resulting in long-term metabolic, neuronal and behavioural changes (Olfe e t a l . , 2 0 1 0 ). Upon arrival, animals were immediately placed into home cages and treated in a manner identical to in-house bred animals. No stress groups (n =11) were bred in-house at the CCBN.  Gradually, the frequency of the bars was increased until the animal could not differentiate the bars and was designated a failed trial. The frequency was then decreased with every failed trial until the animal could differentiate the bars once again. This was performed in the clockwise (CW) and counterclockwise (CCW) direction, randomly alternating directions for each trial, and continued until a frequency threshold (visual acuity) for the animal was found (Alam e t a l . , 2 0 2 2 ).

EAE Induction and Symptom Monitoring
It is important to note that the term acuity in the present study refers to the maximum spatial frequency that evokes an optomotor response. The animal was then returned to its home cage.
During the course of EAE, optic neuritis was hypothesized to cause severe visual acuity impairments. As such, a maximum of thirty trials was designated based on baseline measurements of the frequency thresholds; typically, a total of 15-20 trials per rotations is required to measure a contrast sensitivity function through each eye. Once this maximum was reached, the last recorded threshold was saved.

Locomotor Activity and Exploration Task
An AccuScan activity monitoring system (AccuScan Instruments Inc., OH, USA) with

Blood Collection and Spleen Weights
Baseline blood samples were collected 7 days prior to EAE induction at P43 and during the peak of symptoms at 19 days post-induction (day19). Briefly, in manually restrained mice, a small puncture was made in the sub-mandibular vein using a lancet, and approximately 0.1 ml of blood was collected with a micro-tube. All samples were collected in the morning hours between 09:00 and 11:00 h. Animals were returned to their home cage and allowed to recover for 4 days.
Post-recovery blood samples were collected immediately prior to euthanization (day27) through a cardiac puncture using a heparin coated syringe. Animals were euthanized through an intracardiac injection of Euthansol (pentobarbital, 150 mg/kg; CDMV Inc., Saint-Hyacinthe, QC, Canada). Plasma was obtained by centrifugation at 5,000 rpm for 10 min and stored at −80°C.
Spleen was removed and weighed on an electronic scale to determine the possible impact of ELS and EAE on the organ central to endocrine and immune regulation (Faraji e t a l . , 2 0 2 2 a ).

Data Analysis
In all statistical analyses (SPSS 16.0, SPSS Inc., USA), a p-value of <0.05 (two-tailed) was chosen as the significance level, and results are presented as mean ± standard error. EAE disease scores, optokinetic responses and CORT levels were often not normally distributed, and Levene's test did not confirm homogeneity of variance in the spleen weights. Therefore,

Declaration of Competing Interest
The authors declare no potential conflict of interest.

Author contributions
DB and GASM designed the study. DB and JF performed the experiments and analyzed the data. JF wrote the paper. DB, VWY and GASM edited the paper. VWY and GASM acquired the funding and provided resources.      e  n  c  e  p  h  a  l  o  m  y  e  l  i  t  i  s  -o  p  t  i  c  n  e  u  r  i  t  i  s  .   J  N  e  u  r  o  i  n  f  l  a  m  m  a  t  i  o  n  ,  1  7   (  1  )  ,  2  1  6  .  h  t  t  p  s  :  /  /  d  o  i  .  o  r  g  /  1  0  .  1  1  8  6  /  s  1  2  9  7  4  -0  2  0  -0  1  8  8  9  -z   H  e  r  n  a  n  d  e  z  ,  M  .  E  .  ,  M  a  r  t  i  n  e  z  -M  o  t  a  ,  L  .  ,  S  a  l  i  n  a  s  ,  C  .  ,  M  a  r  q  u  e  z  -V  e  l  a  s  c  o  ,  R  .  ,  H  e  r  n  a  n  d  e  z  -C  h  a  n  ,  N  .  G  .  ,  M  o  r  a  l  e  s  -M  o  n  t  o  r  ,  J  .  ,  P  é  r  e  z  -T  a  p  i i  o  r  e  n  a  l  m  e  t  a  b  o  l  i  c  b  i  o  m  a  r  k  e  r  s  l  i  n  k  e  a  r  l  y  l  i  f  e  s  t  r  e  s  s  t  o  r  i  s  k  o  f  n  o  n  -c  o  m  m  u  n  i  c  a  b  l  e  d  i  s  e  a  s  e  s  a  n  d  a  d  v  e  r  s  e  m  e  n  t  a  l  h  e  a  l  t  h  o  u  t  c  o  m  e  s  .   S  c  i  R  e  p  ,  1  0 (