An Intravenous Injection of Reelin Rescues Endogenous Reelin Expression and Epithelial Cell Apoptosis in the Small Intestine Following Chronic Stress

Abstract

Chronic stress disrupts the integrity of the gut environment, including leaking of the intestinal epithelium. Reelin, an extracellular matrix protein, is released from cells of the lamina propria and promotes epithelial cell proliferation and migration up the crypt-villus axis to facilitate renewal of the gut lining. In the present study, we evaluated Reelin expression and apoptosis in the small intestine of Long Evan's rats treated with recombinant Reelin (3 µg) or vehicle following 3 weeks of daily corticosterone (40 mg/kg/day) or vehicle injections. We show that Reelin- and cleaved caspase-3- immunoreactive cells are diminished in the lamina propria or epithelial cells of the gut lining following chronic stress (∼ 50% and 55%, respectively), and that a single injection of 3 µg of Reelin delivered intravenously can reverse these parameters. We also found Reelin cell counts in the small intestine did not correlate to counts in the hippocampus regardless of exposure to chronic stress or Reelin treatment. Our results suggest that Reelin may serve a protective function over gut barrier integrity through the restoration of epithelial cell turnover, and that Reelin may have a role in reversing chronic stress-induced changes to the gut environment.

Keywords

chronic stress leaky gut depression corticosterone gut-brain axis immunohistochemistry reelin apoptosis crypt-villus axis intestinal lining regeneration

Introduction

Under healthy conditions, the gut maintains selective control over translocation of nutrients from the gut into circulation.¹ The gut barrier can become more permeable, or “leaky” following exposure to chronic stress and in stress-related conditions such as major depressive disorder (MDD).^2,3 Glucocorticoids bind to cytoplasmic glucocorticoid-α receptors (GRαs) in gut epithelial cells to enhance tight junction function and inhibit tumor necrosis factor-α (TNF-α)-induced increases in intestinal barrier permeability, serving a protective role over leaky gut.⁴ Upon glucocorticoid binding to GRs, the glucocorticoid-GR complex rapidly translocates to the nucleus where it inhibits myosin light chain kinase (MLCK) promotor activity, an established component of TNF-α-induced disruption of tight junction proteins.⁴ When glucocorticoids are chronically elevated, however, the opposite effect on gut barrier permeability is observed.⁵ Intestinal lining permeability is additionally affected by mineralocorticoid receptors, where a blockade of mineralocorticoid receptors decreases the expression of claudins, primary regulators of intestinal permeability.⁶ Furthermore, immune cells become resistant to the immunosuppressive effects of glucocorticoids in chronic stress, contributing to an increased release of pro-inflammatory factors that can further damage cells of the gut barrier and disrupt tight-junction function.⁷ Increased permeability of the gut barrier can allow harmful bacteria, toxins, and metabolites to enter circulation, which can contribute to the development of depression.⁸ Therefore, treatments that restore gut barrier integrity may serve a prophylactic function by supporting resistance to stress-induced inflammation.^9–11

Reelin is a large pleiotropic extracellular matrix protein present throughout the body, including the brain, liver, intestines, and blood.¹² Reduced hippocampal Reelin-immunoreactive (IR) cell counts and reduced plasma Reelin levels have been shown in individuals diagnosed with MDD.^13,14 Reduced Reelin-IR cell populations in the subgranular zone of the hippocampus have similarly been shown in rodents exposed to chronic stress, a common foundation for animal models of depression.^15–17 Exposure to daily corticosterone (CORT) injections at 40 mg/kg for 21 days reliably induces behavioural and physiological alterations associated with depression and is consistent with depressive-like phenotypes elicited by other established chronic stress paradigms.^18–22 Previous work from our group has shown that a single intracerebroventricular or a single intravenous injection of Reelin can reverse behavioural and physiological deficits associated with chronic CORT exposure,^{15–19,23–25} and that 3 µg of Reelin is the most effective intravenous dose in producing antidepressant-like effects in this model.¹⁵

Reelin is also present in the lamina propria of the gut, where it is thought to promote the migration of intestinal epithelial cells up the crypt-villus axis and regulate gut barrier cell turnover.²⁶ The gut lining is renewed every 3–5 days, an important process to ensure that the cells comprising the gut barrier are healthy, functioning, and intact.²⁷ Mice heterozygous for a Reelin gene mutation (in the form of a partial deletion of approximately 150 kb) express ∼50% less endogenous Reelin and present with reduced cell proliferation in intestinal crypts and slower cell migration up villi, indicating a role for Reelin in maintaining proper gut barrier function.²⁸

In the present study, we evaluated whether chronic stress brings about alterations to intestinal Reelin immunoreactivity and apoptosis in small intestine villi, and if a single intravenous injection of 3 µg of Reelin can normalize these parameters. Secondarily, we evaluated if these markers correlate with neurobiological markers (hippocampal Reelin-IR and GluA1-IR cell counts) found in the same rats following chronic stress and Reelin treatment.¹⁵ Given previous research showing decreases in Reelin levels in chronic stress and MDD, and Reelin's role in promoting epithelial cell proliferation and migration in the intestine, we hypothesized that Reelin would be diminished in the gut in chronic stress, that this would decrease gut lining regeneration as indicated by apoptosis at villi tips, and that the administration of 3 µg of Reelin via the lateral tail vein would reverse these alterations.

This study aimed to further elucidate a role for Reelin in the gut, as there are a limited number of studies that that consider intestinal Reelin, especially under conditions of chronic stress. As the gut-brain axis becomes a more prominent field of study in the context of psychiatric disorders, it is important that we further investigate Reelin's functions in the gut environment to build on evidence for Reelin as a necessary protein for maintaining proper functioning of the gut barrier.²⁴

Methods

Animal Husbandry

The samples used for this study come from a subset of rats (N = 32) used in Allen et al, 2022. Male Long Evans rats (Charles River, QC, Canada) were housed individually in rectangular polypropylene cages according to guidelines of the Canadian Council on Animal Care, and approved by the University of Victoria animal care committee. Rats received food and water ad libitum. The room was maintained at 21 °C, and lights were set to a 12-h light/dark cycle.

Chronic Stress Model and Animal Behaviour

Rats were allowed to habituate for 7 days upon arrival, and were assigned to one of four groups (n = 8): vehicle/ vehicle (V/V), vehicle/ Reelin (V/R), CORT/ vehicle (C/V), or CORT/ Reelin (C/R). Rats received subcutaneous injections of either vehicle (V/V and V/R groups; 0.9% NaCl with 2% polysorbate-80), or CORT (C/V and C/R groups; 40 mg/ kg suspended in vehicle, Steraloids) at a volume of 1 ml/kg daily for 21 days. On the last day of treatment, sterile 0.1 M phosphate-buffered saline (PBS) or recombinant Reelin (3μg for C/R, 9 μg for V/R, dissolved in 0.1 M PBS) was administered via the lateral tail vein. A higher dose of Reelin was used in control animals to investigate any potential adverse reactions. The recombinant Reelin protein (3820-MR-025-CF, R&D Systems) is composed of Reelin repeats 3-6, the central fragment of the Reelin protein.

Rats underwent behavioural tests as outlined in Allen et al, 2022.¹⁵ Briefly, rats performed the forced swim test (FST; 10 min with a water temperature of 27 ± 2 °C), the open field test (OFT; 5 min), and the object-in-place test (10 min habituation followed by 5 min sample phase with four different objects, and a test phase of 5 min to explore the four objects with two objects in different locations). Please refer to Allen et al, 2022 for detailed behavioural methodology and results (Figure 1).

Perfusions, Tissue Collection, and Tissue Storage

Rats were deeply anaesthetized using 5% isoflurane before being perfused transcardially with ice-cold 0.9% NaCl followed by ice-cold paraformaldehyde (PFA; 4% w/v in 0.1 M PB). A middle segment of the small intestine was removed and postfixed in 4% PFA for 48 h at 4 °C. Intestinal segments were then transferred to increasing concentrations of sucrose solution (10%, 20%, and 30% w/v) every 24 h. Segments were stored in a 30% (w/v) sucrose solution containing 0.1% (w/v) sodium azide until the time of the study.

Tissue Processing

Intestinal segments of approximately 1 cm in length were opened longitudinally, washed in TBS, and frozen in optimal cutting temperature compound (OCT)-filled embedding molds using liquid nitrogen. Tissue was sliced with a cryostat (Vibratome ULTRAPRO 5000) at a thickness of 20 μm and mounted on Super-Frost Plus Microscope glass slides for immunohistochemical analyses.

Immunohistochemistry

Reelin

Reelin-positive cells were visualized using 3,3’-diaminobenzidine (DAB)-immunohistochemistry (Figures 1 and 2). First, an antigen retrieval step was performed by incubating slides in 0.175 M sodium citrate at 90 °C for 15 min. Slides were washed in 0.1 M tris-buffered saline (TBS), and treated with 3% hydrogen peroxide (H₂O₂) in TBS for 15 min. Slides were blocked in blocking solution consisting of 1% (w/v) BSA, 15% (v/v) NGS, and 0.3% (v/v) Triton X-100 in 0.1 M TBS for 1 h. Subsequently, slides were incubated with a mouse anti-Reelin primary antibody (Millipore, MAB5364; 1:500 in blocking solution) overnight at 4 °C. Control reactions were carried out without the primary antibody. The next day, slides were incubated at room temperature (RT) for 2 h with a biotinylated goat anti-mouse secondary antibody (Sigma-Aldrich, BA-9200; 1:200 in blocking solution). Finally, slides were incubated with an avidin-biotin complex (Vecta Stain Elite ABC reagent, Vector Laboratories; 1:500), and visualized using 0.002% (w/v) DAB (Sigma-Aldrich) and 0.0078% (v/v) H₂O₂. Slides were dehydrated through a series of increasing ethanol concentrations, cleared with xylenes, and coverslipped using Permount mounting medium (Fisher Scientific SP15-500).

Figure 1.

Experimental Timeline and Immunostaining. Rats Were Injected (s.c.) Daily for 21 Days with Vehicle (0.9% NaCl with 2% Polysorbate-80) or CORT (40 mg/kg, Suspended in Vehicle). On the Last day of Injections, Rats Were Administered Vehicle (0.1 M PBS) or 3 µg of Recombinant Reelin at a Volume of 0.5 mL via the Lateral Tail Vein. Rats Underwent Behavioural Tests and Were Perfused 4 Days post- Reelin Injection. A Middle Segment of the Small Intestine was Cryosectioned at 30 µm, and Cross Sections Were Stained for Reelin or Cleaved Caspase-3. Reelin and Cleaved Caspase-3 Density Were Determined and Compared Between Treatment Groups.

Figure 2.

Reelin-IR Cell Density in the Small Intestine after CORT and/or Reelin Administration. A: Representative Photomicrographs (20x and 63x magnification) of Reelin-IR Cell Populations in Villi of the Rat Small Intestine. B: Density of Reelin-IR Cells for the Different Treatment Conditions. CORT Decreased the Density of Reelin-IR Cells and Reelin Treatment Restored it. All data are Expressed as Mean ± SEM; V/V = vehicle/ vehicle; V/R = vehicle/ Reelin; C/V = CORT/ vehicle; C/R = CORT/ Reelin. *p < 0.05, ** p ≤ 0.01.

Cleaved Caspase-3

Slides were incubated with sodium citrate (0.175 M) at 90 °C for 15 min for antigen retrieval. Slides were treated with 3% hydrogen peroxide in TBS for 30 min before incubation in a blocking solution of 1% (w/v) BSA, 15% (v/v) NGS, and 0.3% (v/v) Triton X-100 in TBS for 2 h. Incubation with the primary antibody against cleaved caspase-3 (Cell Signaling Technology, 9661S; 1:600 in blocking solution) took place for 24 h at 4 °C. The next day, slides were incubated with a secondary goat anti-rabbit IgG antibody (Vector Laboratories, VECTBA1000; 1:200 in blocking solution) for 2 h (RT). Finally, slides were incubated in an avidin-biotin complex (Vecta Stain Elite ABC reagent, Vector Labs; 1:500), and visualized using 0.002% (w/v) DAB (Sigma-Aldrich) and 0.0078% (v/v) H₂O₂. Slides were dehydrated through a series of increasing ethanol concentrations, cleared with xylenes, and coverslipped using Permount mounting medium (Fisher Scientific SP15-500). Results are presented in Figure 3.

Figure 3.

CORT Decreased Apoptotic Cells in the Upper Portion of Small Intestine Villi. Results are Expressed as the Number of Cleaved Caspase-3-IR Cells per 100 Villi. A: Representative Photomicrographs of Cleaved Caspase-3-IR Cells in Villi of the rat Small Intestine for Each Treatment Group (20x and 63x Magnification). B: CORT Administration Significantly Decreased Cleaved Caspase-3-IR Populations, Which was Partially Rescued with Reelin Administration (∼50%). All Data is Expressed as Mean ± SEM; V/V = Vehicle/ Vehicle; V/R = Vehicle/ Reelin; C/V = CORT/ Vehicle; C/R = CORT/ Reelin. *p < .05, ** p ≤ .01.

Quantification of Immunoreactive Cells

Reelin

Reelin-IR cells in the small intestine were visualized with a Zeiss Axioimager M.2 miroscope. The sample was traced at 2.5x magnification along the perimeter of the muscularis externa and around the villi in the interior of the section. Reelin-IR cells were counted using an unbiased optical fractionator method (Neurolucida, 2024.1.3, MBF Bioscience) at 20x magnification.

Quantification was performed as previously described by our lab,¹⁵ with number estimates calculated using the following formula: N total: ΣQ− × 1/ssf × A(x,y step) ÷ a(frame) × t/h, where ΣQ− is the number of counted cells; ssf is the section sampling fraction (1 in 6); A(x,y step) is the area associated with each x,y movement (199,809 μm²); a(frame) is the area of the counting frame (40,000 μm²); t is the weighted average section thickness; and h is the height of the dissector (9 μm). A guard zone of 2 μm was used to avoid counting sectioning artefacts. Results are expressed as the density of Reelin-IR cells (cells/ mm²).

Cleaved Caspase-3

Stained small intestine cross sections were observed using a Zeiss Axioimager M.2. At least 100 villi per animal were marked at 2.5x using Neurolucida (2024.1.3, MBF Bioscience). To avoid bias, the entire last cross section was labelled and counted rather than counting exactly 100 villi per rat. Villi were marked if they were intact and well-oriented longitudinally. Apoptotic cells at villi apices were counted in marked villi at 20x magnification. Results are expressed as the number of cleaved caspase-3-IR cells per 100 villi.

Statistical Analyses

This study used a total of 32 animals for all experiments discussed. Animals were assigned to one of four groups (n = 8), as we had two dependent variables: the treatment over 21 days (vehicle or CORT), and the treatment on day 21 (vehicle or Reelin). Statistical analyses were performed using JASP (JASP Team (2024). JASP (Version 0.95.0)[Computer software]) or GraphPad Prism (version 10.4.2; GraphPad Software, San Diego, CA, USA), and graphs were generated using GraphPad Prism. Data are presented as means ± standard error of the mean (SEM). Data were considered statistically significant if p < 0.05. Treatment group differences in Reelin-IR and cleaved caspase-3-IR cell density were assessed using a two-way analysis of variance (ANOVA) followed by Tukey's HSD multiple comparisons test. We evaluated Pearson correlations between intestinal Reelin-IR cell density or cleaved caspase-3-IR cell counts and neurobiological markers in the brain (Reelin-IR and GluA1-IR cell counts in the hippocampus), and between Reelin-IR cell density and cleaved caspase-3-IR cell counts in the small intestine. Pearson correlations are shown in Table 1 outlining coefficients (r) with 95% confidence intervals and a linear regression provided with the results.

Table 1.

Correlations Between Reelin-IR Cell Density, Caspase-3-IR Cell Counts, and Neurobiological Markers.

Comparison	Group	n	Pearson's r	p-value	95% CI	DFd
Reelin-IR Cell Density (Small Intestine)/ Caspase-3-IR Cell Counts (Small Intestine)	Vehicle/ Vehicle	8	0.4556	.2566	−0.3669, 0.8783	6
	Vehicle/ Reelin	8	0.08641	0.8388	−0.6584, 0.7457	6
	CORT/ Vehicle	8	0.2613	0.5319	−0.5435, 0.8158	6
	CORT/ Reelin	8	−0.2051	0.6261	−0.7949, 0.5840	6
Reelin-IR Cell Density (Small Intestine)/ Reelin-IR Cell Counts (Hippocampus)	Vehicle/ Vehicle	8	0.2684	0.5205	−0.5381, 0.8183	6
	CORT/ Vehicle	8	0.2827	0.4976	−0.5270, 0.8233	6
	CORT/ Reelin	8	−0.5479	0.1598	−0.9037, 0.2554	6
Caspase-3-IR Cell Counts (Small Intestine)/ Reelin-IR Cell Counts (Hippocampus)	Vehicle/ Vehicle	8	0.03308	0.938	−0.6876, 0.7209	6
	CORT/ Vehicle	8	-0.1922	0.6484	−0.7899, 0.5927	6
	CORT/ Reelin	8	0.3235	0.4344	−0.4937, 0.8373	6
Reelin-IR Cell Density (Small Intestine)/ GluA1-IR Cell Counts (Hippocampus)	Vehicle/ Vehicle	8	0.1022	0.8701	−0.8574, 0.9030	6
	Vehicle/ Reelin	8	−0.2214	0.5982	−0.8011, 0.5726	6
	CORT/ Vehicle	8	−0.09045	0.885	−0.9008, 0.8605	6
	CORT/ Reelin	8	−0.5037	0.4963	−0.9870, 0.8866	6
Caspase-3-IR Cell Counts (Small Intestine)/ GluA1-IR Cell Counts (Hippocampus)	Vehicle/ Vehicle	8	0.1017	0.8707	−0.8575, 0.9030	6
	Vehicle/ Reelin	8	−0.5521	0.1559	−0.9048, 0.2497	6
	CORT/ Vehicle	8	−0.5955	0.2894	−0.9688, 0.6042	6
	CORT/ Reelin	8	−0.7381	0.2619	−0.9940, 0.7673	6

A Pearson Correlation with a 95% Confidence Interval was Used to Assess the Linear Relationship Between Variables. the Table Outlines Sample Size (n), the Correlation Coefficients (r), p-Values, Confidence Intervals, ad the Degrees of Freedom All Data is Expressed as Mean ± SEM; *p < .05, ** p ≤ .01.

Results

Reelin Rescues CORT-Induced Deficits in Reelin Immunoreactive Cell Density in the Small Intestine

A two-way ANOVA was revealed significant main effects of chronic CORT administration (F_{(1, 28)} = 5.86, p = .022, ω² = 0.103), and Reelin injection (F_{(1, 28)} = 7.67, p = .010, ω² = .141) (Figure 2A and B). There was a significant interaction between CORT and Reelin treatment (F_{(1, 28)} = 4.82, p = 0.037, ω² = 0.081). After conducting Tukey's post hoc test, we observed a significant decrease in Reelin-IR cell density in rats that received daily CORT in the absence of Reelin administration (p = .014). Reelin-IR cell density was normalized in CORT-treated rats following the administration of 3 µg of recombinant Reelin (p = 0.08).

Cleaved Caspase-3 Immunoreactive Cell Counts Were Decreased Following Chronic Stress and Were Partially Recovered with Reelin Administration

A two-way ANOVA was conducted to determine if treatment with CORT or Reelin affected caspase-3-IR cell prevalence in small intestine villi. There was no significant main effect for either CORT (F_{(1, 28)} = 2.827, p = .104, ω² = 0.046) or Reelin (F_{(1, 28)} = 0.229, p = .636, ω² = 0.000) administration. There was a statistically significant interaction between CORT and Reelin administration on caspase-3-IR cell density, F_{(1, 28)} = 7.455, p = .011, ω² = 0.163. Post hoc analyses using Tukey's multiple comparisons test revealed a significant difference in caspase-3-IR cell density in rats that received CORT without Reelin (C/V group) compared to rats that received vehicle (V/V group), p = .02. In CORT-treated rats, Reelin recovered caspase-3–IR cell density by approximately 55% compared to CORT/vehicle controls.

No Significant Correlations Between Reelin Cell Density, Cleaved Caspase-3 Cell Counts, and Neurobiological Markers

Correlation analyses showed no significant correlations between Reelin-IR cell density and cleaved caspase-3 cell counts in the small intestine for any treatment group (Table 1, comparison 1). Similarly, no significant correlations were found between Reelin and cleaved caspase-3 in the small intestine and neurobiological markers (Reelin-IR cell counts and the AMPA receptor subunit GluA1 in the hippocampus) quantified in the same animals used in this experiment in Allen et al, 2022 (Table 1, comparisons 2-5). Statistics are outlined in Table 1.

Behavioural Results from Allen et al, 2022

For ease of reading, we have included a brief overview of the behavioural results for these rats previously published in Allen et al, 2022.¹⁵ In the forced swim test, 21 days of CORT significantly increased immobility, and a single injection of 3 µg of Reelin following chronic CORT significantly decreased immobility (by 55%). CORT treated animals had significantly lower discrimination ratios in the object-in-place recognition test as compared to vehicle treated animals, and Reelin partially recovered CORT-induced deficits in discrimination ratio, indicating that Reelin improves hippocampal-dependent cognition. No group differences were found in the OFT.

Discussion

This study showed that chronic stress in the form of repeated CORT injections disrupts the density of Reelin-IR cells and decreases apoptosis in the small intestine, and that these alterations can be reversed with one intravenous injection of 3 µg of recombinant Reelin. We present intestinal Reelin as an integral component of a healthy gut environment, primarily due to its influence over gut barrier renewal. Slowed epithelial cell replacement due to chronic stress may contribute to increased intestinal permeability as cells are constantly damaged by physical, chemical, microbial, and inflammatory agents present in the gut.

We showed that Reelin-IR cell populations are diminished in the small intestine following chronic stress, parallelling previous studies in our lab investigating Reelin expression in the hippocampus,^15–17,29 and hypothalamus.²³ This decrease in Reelin may be in part due to the relationship between chronic stress, branched-chain amino acids (BCAAs), and mTOR signalling. mTOR is a regulatory protein that is activated by Reelin signalling that controls protein synthesis involved in synapse formation, memory, and plasticity. In cases of chronic stress, levels of BCAAs can increase, which is linked to the body's attempt to mediate the effects of stress and promote stress resilience.³⁰ BCAAs, particularly leucine, play a significant role in activating the mTOR signalling pathway, and when mTOR is overactive, it can impair Reelin's signalling.^31–33 As Reelin controls gut lining renewal, it is possible that chronic CORT disrupts gut barrier integrity at least partly through the slowing of cell migration up the crypt villus axis; a necessary process to maintain the gut barrier. As previously explained, continuous cell turnover ensures that damaged or ineffective cells are rapidly replaced, thereby maintaining a gut barrier composed of healthy and functional cells. It is not uncommon for individuals with depression to present with leaky gut,^34,35 which can exacerbate symptoms through immune reactions to leaked bacterial metabolites, antigens, and toxins from the gut lumen into the bloodstream.⁸ Reelin-IR cell populations were restored with the administration of 3 µg of recombinant Reelin. If Reelin restored normal function of the gut barrier, this may have reduced excessive BCAA absorption to decrease detrimental effects on the overactivation of mTOR and restored Reelin levels to homeostatic production. Considering Reelin is necessary for gut lining renewal, and the administration of 3 µg of recombinant Reelin rescued Reelin cell populations in the small intestine following chronic stress, we suggest that Reelin administration may be able to ameliorate stress-induced alterations to gut lining health and function.

Apoptosis occurs in the upper portions of villi in cells that have been pushed upwards by the migration of younger cells up the crypt-villus axis and subsequently lose their attachment to the extracellular matrix and neighbouring cells to be ultimately shed into the lumen of the gut.³⁶ This process is essential to ensuring that the gut barrier is comprised of healthy and functioning cells and minimizes the extent of cellular damage in the gut barrier due to the exposure to various insults from the lumen. Caspase-3 is a key executioner protease in apoptosis and is cleaved by initiator caspases (caspase-8-, and −9) to activate procaspase-3 to cleaved caspase-3.³⁷ Once activated, caspase-3 orchestrates the dismantling of the cell through the cleavage of various substrates. Chronic stress induced by repeated CORT decreased cleaved caspase-3-IR cells at villi tips, suggesting that chronic stress slows epithelial cell turnover. Apoptosis was partially restored (∼50%) following Reelin administration, suggesting that Reelin may increase epithelial cell migration to encourage gut lining turnover, thereby acting as a protective factor for gut lining integrity. This could have implications for the treatment of gastrointestinal conditions that involve a leaky gut or susceptibility to a damaged gut barrier and could also inform approaches to managing depression that co-occurs with gastrointestinal symptoms. Estimates indicate that approximately 70% of individuals who experience depressive episodes have concomitant gastrointestinal issues, and that these gastrointestinal symptoms are associated with increased symptom severity.³⁸ Similarly, individuals with gastrointestinal conditions are more likely to experience symptoms of depression and anxiety.^39–42

We found no significant correlations between Reelin-IR cell density or cleaved caspase-3-IR cell counts in the small intestine and neurobiological markers evaluated in the same rats in Allen et al, 2022, or between Reelin-IR cell density and cleaved caspase-3-IR cell counts in the small intestine. It is likely that the high dose of CORT was sufficient to minimalize individual variability between rats in the C/V and C/R treatment groups in terms of Reelin and cleaved caspase-3 levels. Although we do see more individual variability in the V/V rats and the V/R rats, lower Reelin does not significantly correlate with lower cleaved caspase-3 for individual rats. This may be because, despite some rats having lower Reelin levels than others, the levels remain sufficient to support its role in promoting epithelial cell migration up the crypt-villus axis. Intestinal Reelin and cleaved caspase-3 expression did not correlate significantly with neurobiological markers. While there is clear evidence for the gut-brain connection, most of this research focuses on small messengers like short-chain fatty acids, hormones, and neurotransmitters as mediators. If lower levels of Reelin contributed to leaking of the intestinal epithelium in vehicle rats, the resulting increase in permeability may not have been severe enough to trigger inflammation that influenced the neurobiological markers measured, and thus no correlation was observed. Furthermore, although the gut and brain communicate and influence each other, they are distinct systems, and reduced Reelin production in the gut does not necessarily reflect reduced Reelin levels in other regions of the body for a given rat.

MDD and chronic stress are associated with increased intestinal lining permeability, and individuals with MDD or under conditions of chronic stress are at an increased risk for gastrointestinal conditions such as irritable bowel syndrome.^38–42 Repeated injections of CORT over 21 days replicates many symptoms and markers associated with MDD, including increased despair-like behaviour as assessed by the forced swim test, decreased Reelin-IR cell density in the hippocampus, alterations to serotonin transporter clustering patterns on blood lymphocytes, and imbalances in glutamatergic and GABAergic neurotransmission.^{15–18,43–46} Furthermore, chronic CORT atrophies spleen white pulp, which primarily consists of lymphocytes.^24,25 A single 3 µg injection of recombinant Reelin via the lateral tail vein is sufficient to normalize these parameters. The present study extends previous research from our lab by investigating alterations to the gut environment following treatment with chronic CORT and Reelin. We show that chronic CORT decreases intestinal Reelin expression and reduces apoptosis at villi tips, and that 3 µg of recombinant Reelin normalizes Reelin expression in the lamina propria and increases epithelial cell apoptosis at villi tips. As previous research has placed Reelin as a necessary factor for proper renewal of the gut lining,²⁸ our results suggest that chronic stress may disrupt gut lining renewal through the diminishment of Reelin expression in cells underlying the gut epithelium, and that the administration of recombinant Reelin can ameliorate dysfunction of the gut lining.

Overall, the present study suggests that chronic stress decreases intestinal Reelin and indicates Reelin as a mediator of gut barrier renewal. These findings have putative important implications for management of MDD, particularly in individuals concomitant for gastrointestinal conditions. If Reelin protects against leaky gut through the promotion of gut barrier turnover, Reelin may thereby protect against the exacerbation of depression symptoms triggered by inflammatory immune responses.

Footnotes

ORCID iDs

Ciara S. Halvorson

Brady S Reive

Josh Allen

Lisa E Kalynchuk

Hector J Caruncho

Ethical considerations

All procedures were approved by the University of Victoria animal care committee, and were carried out according to the guidelines of the Canadian Council on Animal Care.

Consent to Participate

Not applicable.

Consent for Publication

Not applicable.

Author Contributions

CSH: Contributed to experimental design, including selection of staining targets, contributed to the development of the staining protocols, cryosectioned tissue, stained tissue, analyzed density of Reelin-IR and cleaved caspase-3-IR cells, prepared the manuscript.

CLSL: Contributed to the development of staining protocols, contributed to the preparation of the manuscript.

BSR: Assisted with imaging and cell density quantification, contributed to statistical analyses, and supported manuscript preparation.

LSS: Assisted with cryosectioning, counted Reelin and caspase-3-immunoreactive cells for the analysis of density.

JA: Generated the tissue- carried out the injections of CORT and Reelin, behavioural assessments, and harvested the small intestine tissue.

HJC and LEK: Conceptualized the project, provided supervision of the project, contributed to experimental design and interpretation of results, and guided the writing and revision of the manuscript.

Funding

This work was supported by the NSERC Canada Graduate Scholarships- Master's Program (CGS-M) to CSH, NSERC DG to HJC and LEK, and CIHR PG and CRC (HJC)

Canadian Institutes of Health Research, Natural Sciences and Engineering Research Council of Canada, (grant number Canada Research Chair, Project Grant, CGSM, Discovery Grant).

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

References

Dmytriv

Storey

Lushchak

. Intestinal barrier permeability: The influence of gut microbiota, nutrition, and exercise. Front Physiol. 2024 Jul 8;15:1380713. doi:10.3389/fphys.2024.1380713

Cho

Park

Lim

, et al Anxiety, depression and quality of life in patients with irritable bowel syndrome. Gut Liver. 2011 Mar;5(1):29–36. doi:10.5009/gnl.2011.5.1.29

Halvorson

Sánchez-Lafuente

Johnston

, et al Molecular mechanisms of reelin in the enteric nervous system and the Microbiota-gut-brain axis: Implications for depression and antidepressant therapy. Int J Mol Sci. 2024 Jan 9;25(2):814. doi:10.3390/ijms25020814

Boivin

Kennedy

, et al Mechanism of glucocorticoid regulation of the intestinal tight junction barrier. American Journal of Physiology-Gastrointestinal and Liver Physiology. 2007 Feb;292(2):G590–8. doi:10.1152/ajpgi.00252.2006

Zheng

, et al Corticosterone mediates stress-related increased intestinal permeability in a region-specific manner. Neurogastroenterol Motil. 2013 Feb;25(2):e127–39. doi:10.1111/nmo.12066

Karailiev

Hlavacova

Chmelova

, et al Tight junction proteins in the small intestine and prefrontal cortex of female rats exposed to stress of chronic isolation starting early in life. Neurogastroenterol Motil. 2021 Jun;33(6):e14084. doi:10.1111/nmo.14084

Al-Sadi

Boivin

. Mechanism of cytokine modulation of epithelial tight junction barrier. Front Biosci. 2009 Jan 1;14:2765–78. doi:10.2741/3413

Maes

Kubera

Leunis

. The gut-brain barrier in major depression: Intestinal mucosal dysfunction with an increased translocation of LPS from gram negative enterobacteria (leaky gut) plays a role in the inflammatory pathophysiology of depression. Neuro Endocrinol Lett. 2008 Feb;29(1):117–24.

Bistas

Tabet

. The benefits of prebiotics and probiotics on mental health. Cureus. 2023 Aug 9;15(8):e43217.

10.

Felger

Lotrich

. Inflammatory cytokines in depression: Neurobiological mechanisms and therapeutic implications. Neuroscience. 2013 Aug 29;246:199–229. doi:10.1016/j.neuroscience.2013.04.060

11.

Kouba

de Araujo Borba

Borges de Souza

, et al Role of inflammatory mechanisms in Major depressive disorder: From etiology to potential pharmacological targets. Cells. 2024 Feb 28;13(5):423. doi:10.3390/cells13050423

12.

Khialeeva

Carpenter

. Nonneuronal roles for the reelin signaling pathway. Dev Dyn. 2017 Apr;246(4):217–26. doi:10.1002/dvdy.24462

13.

Fatemi

Earle

McMenomy

. Reduction in reelin immunoreactivity in hippocampus of subjects with schizophrenia, bipolar disorder and major depression. Mol Psychiatry. 2000 Nov;5(6):654–63. 571. doi:10.1038/sj.mp.4000783

14.

Jin

Zhang

Jiang

, et al The role of reelin in the pathological mechanism of depression from clinical to rodents. Psychiatry Res. 2022 Nov 1;317:114838. doi:10.1016/j.psychres.2022.114838

15.

Allen

Romay-Tallon

Mitchell

, et al Reelin has antidepressant-like effects after repeated or singular peripheral injections. Neuropharmacology. 2022 Jun 15;211:109043. doi:10.1016/j.neuropharm.2022.109043

16.

Johnston

Allen

Shkolnikov

, et al Reelin rescues behavioral, electrophysiological, and molecular metrics of a chronic stress phenotype in a similar manner to ketamine. eNeuro. 2023 Aug;10(8):ENEURO.0106–23.2023. doi:10.1523/ENEURO.0106-23.2023

17.

Scheil

KKA

Sánchez-Lafuente

Reive

, et al Time-dependent antidepressant-like effects of reelin and ketamine in the repeated-corticosterone model of chronic stress. Prog Neuropsychopharmacol Biol Psychiatry. 2024 Jun 8;132:110998. doi:10.1016/j.pnpbp.2024.110998

18.

Brymer

Johnston

Botterill

, et al Fast-acting antidepressant-like effects of reelin evaluated in the repeated-corticosterone chronic stress paradigm. Neuropsychopharmacology. 2020 Sep;45(10):1707–16. doi:10.1038/s41386-020-0609-z

19.

Lebedeva

Allen

Kulhawy

, et al Cyclical administration of corticosterone results in aggravation of depression-like behaviors and accompanying downregulations in reelin in an animal model of chronic stress relevant to human recurrent depression. Physiol Behav. 2020 Oct 1;224:113070. doi:10.1016/j.physbeh.2020.113070

20.

Liu

Zhou

Yang

, et al Social defeat stress causes depression-like behavior with metabolite changes in the prefrontal cortex of rats. PLOS ONE. 2017 Apr 28;12(4):e0176725.

21.

You

Luo

Zhang

, et al Pro- and anti-inflammatory cytokines expression in rat’s brain and spleen exposed to chronic mild stress: Involvement in depression. Behav Brain Res. 2011 Nov 20;225(1):135–41. doi:10.1016/j.bbr.2011.07.006

22.

Hui

Zhang

Liu

Zhang

Teng

, et al Learning and memory alterations are associated with hippocampal N-acetylaspartate in a rat model of depression as measured by 1H-MRS. PLoS One. 2011 Dec 14;6(12):e28686. doi:10.1371/journal.pone.0028686

23.

Liria Sánchez-Lafuente

Martinez-Verbo

Johnston

, et al Chronic corticosterone exposure in rats induces sex-specific alterations in hypothalamic reelin fragments, MeCP2, and DNMT3a protein levels. Neurosci Lett. 2024 May 1;830:137770. doi:10.1016/j.neulet.2024.137770

24.

Reive

Johnston

Sánchez-Lafuente

, et al Intravenous reelin treatment rescues atrophy of spleen white pulp and correlates to rescue of forced swim test immobility and neurochemical alterations induced by chronic stress. Chronic Stress (Thousand Oaks). 2023;7:24705470231164920.

25.

Reive

Johnston

Sánchez-Lafuente

, et al Intravenous reelin rescues despair-like behavior, reelin cells in the dentate sub-granular zone, and spleen atrophy in the cyclic corticosterone model of recurring depressive episodes. Front Pharmacol. 2024;15:1368620.

26.

García-Miranda

Vázquez-Carretero

Sesma

, et al Reelin is involved in the crypt-villus unit homeostasis. Tissue Eng Part A. 2013 Jan;19(1-2):188–98. doi:10.1089/ten.tea.2012.0050

27.

Barker

. Adult intestinal stem cells: Critical drivers of epithelial homeostasis and regeneration. Nat Rev Mol Cell Biol. 2014 Jan;15(1):19–33. doi:10.1038/nrm3721

28.

Carvajal

Serrano-Morales

Vázquez-Carretero

, et al Reelin protects from colon pathology by maintaining the intestinal barrier integrity and repressing tumorigenic genes. Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease. 2017 Sep 1;1863(9):2126–34. doi:10.1016/j.bbadis.2017.05.026

29.

Johnston

Thacker

Desjardins

, et al Ketamine rescues hippocampal reelin expression and synaptic markers in the repeated-corticosterone chronic stress paradigm. Front Pharmacol. 2020 Sep 2;11:559627. doi:10.3389/fphar.2020.559627

30.

Nasrallah

Haidar

Stephan

, et al Branched-chain amino acids mediate resilience to chronic social defeat stress by activating BDNF/TRKB signaling. Neurobiol Stress. 2019 Nov;11:100170. doi:10.1016/j.ynstr.2019.100170

31.

Kimball

. Integration of signals generated by nutrients, hormones, and exercise in skeletal muscle1234. Am J Clin Nutr. 2014 Jan 1;99(1):237S–242S. doi:10.3945/ajcn.113.068387

32.

Moberg

Apró

Ekblom

, et al Activation of mTORC1 by leucine is potentiated by branched-chain amino acids and even more so by essential amino acids following resistance exercise. American Journal of Physiology-Cell Physiology. 2016 Jun;310(11):C874–84. doi:10.1152/ajpcell.00374.2015

33.

Teles e Silva

Glaser

Griesi-Oliveira

, et al Rare CACNA1H and RELN variants interact through mTORC1 pathway in oligogenic autism spectrum disorder. Transl Psychiatry. 2022 Jun 6;12:234. doi:10.1038/s41398-022-01997-9

34.

Ohlsson

Gustafsson

Lavant

, et al Leaky gut biomarkers in depression and suicidal behavior. Acta Psychiatr Scand. 2019 Feb;139(2):185–93. doi:10.1111/acps.12978

35.

Slyepchenko

Maes

Jacka

, et al Gut Microbiota, bacterial translocation, and interactions with diet: Pathophysiological links between Major depressive disorder and non-communicable medical comorbidities. Psychother Psychosom. 2017;86(1):31–46.

36.

Andrade

Rosenblatt

. Apoptotic regulation of epithelial cellular extrusion. Apoptosis. 2011 May;16(5):491–501. doi:10.1007/s10495-011-0587-z

37.

Negroni

Cucchiara

Stronati

. Apoptosis, necrosis, and necroptosis in the gut and intestinal homeostasis. Mediators Inflamm. 2015;2015:250762.

38.

Huang

Cai

, et al Gastrointestinal symptoms during depressive episodes in 3256 patients with Major depressive disorders: Findings from the NSSD. J Affect Disord. 2021 May 1;286:27–32. doi:10.1016/j.jad.2021.02.039

39.

Eustis

McCall

Murphy

, et al Association between gastrointestinal symptoms and depression in a representative sample of adults in the United States: Findings from national health and nutrition examination survey (2005–2016). Journal of the Academy of Consultation-Liaison Psychiatry. 2022 May 1;63(3):268–79. doi:10.1016/j.jaclp.2021.08.008

40.

Zhang

Wang

Huang

, et al Prevalence of depression and anxiety in patients with chronic digestive system diseases: A multicenter epidemiological study. World J Gastroenterol. 2016 Nov 14;22(42):9437–44. doi:10.3748/wjg.v22.i42.9437

41.

Zamani

Alizadeh-Tabari

Zamani

. Systematic review with meta-analysis: The prevalence of anxiety and depression in patients with irritable bowel syndrome. Aliment Pharmacol Ther. 2019 Jul;50(2):132–43. doi:10.1111/apt.15325

42.

Fond

Loundou

Hamdani

, et al Anxiety and depression comorbidities in irritable bowel syndrome (IBS): A systematic review and meta-analysis. Eur Arch Psychiatry Clin Neurosci. 2014 Dec;264(8):651–60. doi:10.1007/s00406-014-0502-z

43.

Sánchez-Lafuente

Johnston

Reive

, et al A single intravenous reelin injection restores corticosterone-induced neurochemical and behavioral alterations in dams during the post-partum period. Front Mol Neurosci. 2024;17:1442332.

44.

Rivera-Baltanas

Olivares

Calado-Otero

, et al Serotonin transporter clustering in blood lymphocytes as a putative biomarker of therapeutic efficacy in major depressive disorder. J Affect Disord. 2012 Mar;137(1-3):46–55. doi:10.1016/j.jad.2011.12.041

45.

Rivera-Baltanas

Olivares

Martinez-Villamarin

, et al Serotonin 2A receptor clustering in peripheral lymphocytes is altered in major depression and may be a biomarker of therapeutic efficacy. J Affect Disord. 2014 Jul 1;163:47–55. doi:10.1016/j.jad.2014.03.011

46.

Romay-Tallon

Kulhawy

Brymer

, et al Changes in membrane protein clustering in peripheral lymphocytes in an animal model of depression parallel those observed in naïve depression patients: Implications for the development of novel biomarkers of depression. Front Pharmacol. 2018;9:1149.