Hypothalamic neurochemical changes in long-term recovered bilateral subdiaphragmatic vagotomized rats

doi:10.21203/rs.3.rs-955147/v1

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Hypothalamic neurochemical changes in long-term recovered bilateral subdiaphragmatic vagotomized rats

https://doi.org/10.21203/rs.3.rs-955147/v1

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Background

Vagus nerve is one of the crucial routes in communication between immune and central nervous systems. Impaired vagal nerve function may intensify peripheral inflammatory processes. This effect subsides along with prolonged recovery after permanent nerve injury. One of the results of such compensation is a normalized plasma concentration of stress hormone corticosterone – a marker of hypothalamic-pituitary-adrenal (HPA) axis activity. In the present work we strive to explain this corticosterone normalization by studying the mechanisms responsible for compensation-related neurochemical alterations in the hypothalamus.

Methods

Using microarrays and HPLC we measured genome-wide gene expression and major amino acid neurotransmitters content in hypothalamus of bilaterally vagotomized rats, one month after surgery.

Results

Our results show that, in the long-term, vagotomy affects hypothalamic amino acids concentration but not mRNA expression of tested genes.

Conclusions

We propose an alternative pathway of immune to CNS communication after vagotomy, leading to activation of HPA axis, by influencing central amino acids and subsequent monoaminergic neurotransmission.

Neurobiology of Disease

Hypothalamus

subdiaphragmatic vagotomy

amino acid neurotransmitters

glutamate

GABA

tyrosine

tryptophan

inflammatory response

microarray

HPA axis

Vagus nerve regulates peripheral immune functions in part via HPA axis:

Vagus nerve, due to its extensive peripheral projection range, is a prominent interoceptive pathway [1]. Vagus nerve transmits intraperitoneal immune signals to the CNS [2, 3, 4, 5], causing neurotransmission alterations [6, 7, 8], behavioral changes [8, 9, 10] and indirect activation of hypothalamus [11] - a first element of hypothalamic-pituitary-adrenal (HPA) axis of stress (Figure 1.). HPA activation results in secretion of glucocorticoids (GCs), which are important regulators of the immune system [12]. Moreover, the parasympathetic part of the vagus nerve can directly influence the activity of immune cells in internal organs exposed to pathogens through the cholinergic anti-inflammatory pathway [13, 14, 15].

The physiological tonic activity of vagus nerve (so-called vagal tone) is crucial for maintaining body allostasis. Vagal tone is a critical regulator of the inflammatory response. Low vagal tone results in increased basal plasmatic cortisol, TNF-α, and epinephrine levels, which Bonaz et al. [16] summarized as a pro-inflammatory effect [16, 17]. Vagal tone is also involved in regulating emotional and behavioral responsiveness as well as sickness behaviors - a natural response to ongoing inflammation, which supports the immune system in its fight against the pathogen [18, 19]. Experimental model of abolished vagal tone in context of peritoneal vagal functions is a full subdiaphragmatic truncal vagotomy, which we used in the current experiment.

Vagal dysfunction is compensated for after recovery period:

Vagus nerve integrity is necessary for proper inhibition of inflammatory response. Thus, vagotomy results in highly intensified inflammation and decreased secretion of anti-inflammatory GCs. Surprisingly, these effects diminish after prolonged period of recovery following vagotomy [20, 21]. Our previous results indicate that monoaminergic neurotransmission is altered in hypothalamus after prolonged period of recovery following vagotomy, which might contribute to the restoration of HPA axis activity. These neurotransmission changes may occur due to presence of compensatory mechanisms for impaired immunosensory and anti-inflammatory functions of vagus nerve. Wieczorek and Dunn [11] suggested that function of damaged vagal nerve is substituted by COX-dependent pathway, where immune signals are carried by blood-borne prostaglandin E2 (PGE2). Corroborating this hypothesis, we previously observed that concentration of PGE2 under inflammatory conditions is increased in plasma of vagotomized animals, 30 days after vagotomy procedure [22].

Our aims:

In the current work we investigate, whether transcriptomic changes and/or altered amino acid neurotransmission accompanies monoaminergic alterations in hypothalamus and in consequence may be responsible for preservation of HPA axis activity after recovery from vagotomy. To test those hypotheses we performed a RNA microarray with subsequent RT-qPCR analysis and HPLC concentration analysis of excitatory and inhibitory amino acid neurotransmitters.

Studies were performed on 3-month-old male Wistar rats (300 g ± 25 g, N=75). Animals were individually housed in breeding cages under the following conditions: free access to water and feed (Purina granules), artificial lighting conditions (a 12-hour day-night cycle, light on at 7.00 AM), temperature 21-22°C and 60-65% humidity. Before start of the experiments, the animals were habituated for 7 days to the conditions in the animal facility.

Experiment

Animals were divided into two main groups: sham operated (SH) and subdiaphragmatically vagotomized (VG). Animals in VG group were subjected to surgery under general anesthesia, induced by an intraperitoneal injection of Innovar plus (6 µl/g body weight) and local anesthesia with 2% lidocaine solution (0.5 ml/animal). During operation, a small fragment of gastrointestinal and hepatic branches of the vagal nerve were cut just below the diaphragm. To prevent post-operative infection and to facilitate wound healing an antibacterial agent (Alu Spray, V.M.D.) was applied to the operation site. Bilateral subdiaphragmatic truncal vagotomy procedure and its validation is described in detail in [22]. Sham operation (SH) was performed in analogous way to subdiaphragmatic vagotomy, with exception of cutting the nerves. Following surgeries, the animals were returned to their cages, and after a 30-days-long recovery period, the animals from SH and VG groups were intraperitoneally injected with either saline (0.9% NaCl, 100 µl i.p.) or LPS (10 µg E. coli 026:B6 in 100 µl 0.9% NaCl, 100 µl i.p.). After 120 minutes animals were decapitated. Immediately after decapitation, hypothalamus samples, restricted mainly to the medial part containing the paraventricular nucleus (PVN), were taken.

Samples preparation procedures

For ELISA test of proinflammatory cytokines, plasma was obtained by centrifuging the trunk blood collected on EDTA (1 ml/100 µl of Na₂EDTA), 4 000 rpm for 10 min at 4°C. Obtained plasma was frozen and stored at -80°C until future analysis.

For HPLC analysis of amino acid neurotransmitters, hypothalamuses were immediately homogenized using an ultrasonic homogenizer (BioBlock Scientific, France) for 15 seconds in 150 µl homogenization solution (0.4 mM Na₂S₂O₅, 0.6 mM HClO₄), and centrifuged at 12 000 rpm for 15 minutes at 4°C. At least 100 µl of the supernatant was collected from each sample, transferred to chromatographic tubes and frozen at -80°C.

For microarray and PCR analyses, hypothalamuses (HPTs) were stored in -20°C in 200 µl of RNA-later (Thermo Fisher Scientific, USA). Total RNA was extracted from dissected hypothalamuses using spin column-based Universal RNA Purification Kit (Eurx, Poland) according to the manufacturer's protocol. RNA samples were subjected to DNase treatment on-column (RNase-free DNase I, Eurx, Poland, 15 min, room temperature) as well as in-sample (TURBO DNA-free Kit, Thermo Fisher Scientific, USA, 30 min, 37°C), to remove any residual DNA contamination. The quantity, purity and quality of RNA samples were estimated using a Nanodrop spectrophotometer (Thermo Fisher Scientific, USA) and a Bioanalyzer 2100 microcapillary electrophoresis device (Agilent, USA) with RNA 6000 Nano Assay Kit (Agilent, USA). Samples were of high quality (RNA Integrity Number > 9) and purity (see Supplementary Table S1).

ELISA test for TNF-α

TNF-α concentration was established as a control variable for confirming of ongoing intraperitoneal inflammation. Plasma TNF-α concentration was determined using the Rat TNF-α ELISA Kit, from Diaclone, France (cat. no. 865.000), according to the manufacturer’s instructions. The same statistical assumptions and analytical procedure as for HPLC data was used.

HPLC of amino acids

The concentration of the amino acid neurotransmitters: aspartate (ASP), glutamate (GLU), glycine (GLY), gamma-aminobutyric (GABA), tyrosine (TYR) and tryptophan (TRP) was determined in prepared HPT samples with RP-HPLC-ED gradient method with pre-column derivatization with fresh OPT-thiol reagent (0.1 M Borax, 0.5% OPT, 0.9% mercaptoethanol). The Agilent 1100 chromatographic system with Waters, AccQ Tag for hydrolysate Amino Acid analysis chromatographic column (3.9 x 150 mm) preceded by a Zorbax SB-C18 pre-column (4.6 x 12.5 mm) was used. Column temperature was set at 37°C and mobile phase flow at 0.4 ml/min. The carbon working electrode was set at +0.5 V, relative to the Ag/AgCl reference electrode. The gradient elution was performed using phosphate buffer (5.5 pH) containing: 0.05 M NaH₂PO₄ x H₂O, and addition of methanol; in buffer (A) 20% and (B) 80%. At the start, mobile phase consisted of 100% buffer A. During first 10 min of analysis concentration of buffer B was raised to 10% (in 10th min: 90% A and 10% B). In next 30 minutes content of buffer B in mobile phase was raised from 10–85% (in 40th min: 15% A and 85% B). In next 5 min, content of buffer B was decreased to 0% (in 45th min: 100% A and 0% B). after each sample, system was re-equilibrated for 10 min. The chromatographic data were analyzed using Chemstation, Revision-B.03.02, Agilent software.

HPLC data was tested for normality (Shapiro-Wilk test) and homogeneity of variance (Levene test). For data meeting the assumptions of parametric test ANOVA was used. Others was tested with Kruskall-Walis test with NIR post hoc test. Analogical to analysis performed in our previous work [22], the following two-tailed sub-hypothesis were analyzed and displayed on the figures:

SH+NaCl i.p. vs. VG+NaCl i.p.: Subdiaphragmatic vagotomy does not affect the value of the analyzed variable.
SH+NaCl i.p. vs. SH+LPS i.p.: LPS injection does not affect the value of the analyzed variable under control conditions.
VG+NaCl i.p. vs. VG+LPS i.p.: LPS injection does not affect the value of the analyzed variable in vagotomized animals.
SH+LPS i.p. vs. VG+LPS i.p.: Response to LPS injection is similar in vagotomized and sham operated animals.

P-values lower than 0.05 were considered statistically significant. These statistical analyses were performed using the STATISTICA software, version 13.3 (TIBCO Software Inc. (2017).

Microarray and RealTime qPCR

Microarrays were used to analyze hypothalamic gene expression in all 4 experimental groups. The results of this analysis were validated using real-time qPCR.

Microarray preparation:

21 samples (3-5 per each experimental group, see Supplementary Table S1) were used for microarray analysis. These comprised entire set of samples available in the experiment at that time. Samples were analyzed individually. 100 ng of given sample that passed initial quality control screen (2100 Bioanalyzer, Agilent) were labeled and amplified using GeneChip WT PLUS Reagent Kit and hybridized to an Affymetrix Rat Gene 2.1 ST Array Strip microarray using GeneAtlas Hybridization, Wash, and Stain Kit for WT Array Strips. GeneChip Poly-A RNA Control Kit and GeneChip Hybridization Controls were used to monitor the process of sample preparation. Hybridization to the microarrays was conducted for 20 h at 48°C. Following the hybridization, the microarrays were washed, stained and scanned on a GeneAtlas™ System for Academic Customers. All the reagents and equipment for microarray experiment were provided by Affymetrix, a subsidiary of Thermo Fisher Scientific, USA and the procedures were performed according to manufacturer's protocol. Quality of the analyses was verified using Affymetrix Expression Console Software and standard Affymetrix quality metrics.

All Affymetrix.cel files were firstly imported into Partek® Genomics Suite® software, version 7, build 7.19.1125. (Partek, USA). Prior to any computations, the imported data set was normalized. Background correction was conducted with the use of the RMA method [23] and was followed by quantile normalization. Probes were logged using base 2, while the median polish [24] algorithm was applied to probe set summarization. Definition of all contrasts of interest (comparisons of interest) constituted a base for detection of differentially expressed genes, which was performed with the use of the ANOVA feature of Partek® Genomics Suite®. As a result, every feature’s description contained (among others) p-value for a particular comparison accompanied by its FDR-corrected [24] value and 95% confidence interval for those. Data set was annotated with the Ensembl Transcripts, release 100, genome assembly Rnor_6.0

qPCR analysis:

The results of microarray analysis were validated using SYBR Green-based real-time qPCR. 47 samples (including 21 samples used for microarray experiment, 8-11 samples per each experimental group, see Supplementary Table S1) were used for qPCR analysis. For each sample, 100 ng of total RNA was reverse-transcripted to cDNA using smART RT-PCR Kit (Eurx, Poland). Expression of 7 genes (Actn2, Cxcl14, Gabrg1, Gria1, Oxt, Rab1b, Vim) was studied. Hmbs was used as a reference gene. It was identified as the most stably expressed out of set of candidate reference genes (Gapdh, Hmbs, Hprt1, Tbp, Yhwaz) using the NormFinder tool (see Supplementary Table S3). For details on method of selecting reference gene, see our previous work [25]. For detailed primers description see Supplementary Table S2. Rotor-Gene Q (Qiagen, Netherlands) with Fast SG qPCR Master Mix (2x) (Eurx, Poland) were used to perform qPCR. qPCR reaction conditions were as follows: 1) denaturation - 95°C for 50 s; 2) amplification, 35 cycles - 95°C for 10 s then 10 s in primer-specific annealing temperature then 72°C for 15 s; 3) melting curve analysis - 72-95°C. All assays were executed in triplicate. Each run included five 4-fold serial dilutions for calculating reaction efficiency. A melting curve analysis was performed to verify the presence of one gene-specific peak and the absence of primer-dimer peaks. Pfaffl model was used to calculate relative expression ratios of studied genes. Detailed data on qPCR analysis are presented in Supplementary Table S2. qPCR was performed according to the MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) guidelines [26]. Shapiro-Wilk test was used to test normality of qPCR data. As the data were not distributed normally, Mann–Whitney–Wilcoxon test was used to investigate significance of comparisons, which showed non-adjusted p-value lower than 0.05 in the microarray experiment. As multiple comparisons were made within each analyzed gene, Benjamini-Hochberg method [27] was used to control False Discovery Rate. R script used for statistical analysis of the results can be found in Supplementary Data.

TNF-α-based validation of inflammatory response (Figure 2.)

Increased plasma TNF-α concentration confirms development of inflammatory conditions in both rat groups injected with LPS (H_(3,40)=24.293, p>0.001). However, inflammatory response of vagotomized animals seem to stunted in comparison to sham control (VG NaCl vs. VG LPS p=0.088 compared to SH NaCl vs SH LPS p<0.001), which is in line with observations made by Ghia et al. (2007) [20].

HPLC analysis (Figure 3.)

Statistical analysis revealed significant differences between groups in concentrations of both excitatory amino acid neurotransmitters: 1) ASP (H_3,25=17.68 p<0.001), which concentration under inflammatory conditions increased in vagotomized compared to sham group (VG LPS vs. SH LPS, p=0.006) and 2) GLU (H_3,25=11.66 p=0.008). However, post hoc test of the GLU data did not confirm significance of our assumed sub-hypothesis.

Pronounced differences are visible in case of both inhibitory amino acid neurotransmitters – GLI (H_3,25=12.62 p=0.006) and GABA (F_3,21=19.48 p<0.001). Concentrations of both amino acids were significantly increased after vagotomy (VG NaCl vs. SH NaCl: GLI p=0.038 and GABA p=0.015). GABA concentration was significantly increased after vagotomy also during inflammatory conditions (VG LPS vs. SH LPS, p<0.001; VG LPS vs VG NaCl, p<0.001).

Differences in concentrations of precursors of monoaminergic neurotransmitters were also observed – TYR (F_3,21=6.32 p=0.003) and TRP (H_3,25=18.25 p<0.001). Concentrations of both amino acids were increased after vagotomy in vehicle (VG NaCl vs. SH NaCl; TYR p=0.012, TRP p<0.001) and inflammatory conditions (VG LPS vs. SH LPS; TYR p=0.028, TRP p<0.001). TRP concentration was also increased by LPS injection in both experimental groups; sham and vagotomized (SH LPS vs. SH NaCl; p=0.011; VG LPS vs. VG NaCl; p=0.034).

Microarray experiment (Figure 4.)

Using microarrays, we have analyzed transcriptomic patterns induced by experimental conditions in hypothalamus. None of the observed statistically significant changes in expression of analyzed transcripts survived adjustment for multiple comparisons. Nevertheless, we have selected 7 genes characterized by relatively low non-adjusted p-values in some of the comparisons (mean = 0.019, SD = 0.01, also see Supplementary Table S4), and analyzed their expression using qPCR. The qPCR analysis showed that the studied genes (Actn2, Cxcl14, Gabrg1, Gria1, Oxt, Rab1b, Vim) do not respond to experimental conditions, thus supporting data from microarrays (see Supplementary Table S4).

Vagotomy affects precursors of monoamine neurotransmitters in the hypothalamus

Vagal denervation attenuates inflammation-induced increase of plasma corticosterone and ACTH concentrations for up to two weeks [11, 28]. Afterwards, this effect of vagotomy is diminished. This indicates that proper functioning of anti-inflammatory HPA axis is restored in the long run.

However, even after one month following a vagotomy a monoaminergic neurotransmission in the hypothalamus (a first element of anti-inflammatory HPA axis), as well as in other limbic system structures, is deregulated [22]. We confirmed these results in the current work, by observing an increase in an amount of precursors of the monoaminergic neurotransmitters (tyrosine and tryptophan) in the hypothalamus of the vagotomized animals. In fact, similar tendency to increase tryptophan concentration was also noticed by Wieczorek and Dunn [11] in many brain areas of vagotomized mice. CNS may require higher amounts of the precursors, because they are necessary for reported increased dopamine and serotonin synthesis and turnover in many brain regions after the subdiaphragmatic vagotomy [11, 22]. It is also worth noting that one of the side effects of subdiaphragmatic vagotomy procedure is a slowed movement of food through the digestive system [29], which also may affect the content of tyrosine and tryptophan in blood [30, 31, 32, 33].

Vagotomy affects amino acid neurotransmitters in the hypothalamus

Hypothalamus is innervated by monoaminergic fibers originating from number of brain areas directly or indirectly affected by nucleus tractus solitarius (NTS), which distributes the sensory information from vagus nerve [34, 35, 36, 37, 38]. Those innervations modulates hypothalamic functions i.e. by affecting hypothalamic concentrations of amino acid neurotransmitters (e.g. GABA and Glycine) and, subsequently, post synaptic potentials [39]. Previously, we observed vagotomy-caused changes of monoaminergic neurotransmission in many of those areas (e.g. AM, PAG, HIP) and the hypothalamus itself. In the current work we show a modest increase in the hypothalamic concentration of amino acid neurotransmitters (e.g. GABA and Glycine) in a full recovered animals subjected previously to vagotomy as compared to sham procedure. Similar results were reported by Klarer et al. [40], who studied amino acid neurotransmitters in several other limbic brain structures. Based on these results, we hypothesize that vagotomy-induced changes in hypothalamic amino acid neurotransmitters may be caused by deregulated monoaminergic signaling originating in limbic structures.

In basal conditions, tonic, inter-hypothalamic GABA circuit suppresses the activity of hypothalamic CRF-releasing neurons [41]. Our results show that this mechanism is intensified in vagotomized animals compared to sham-operated animals. We propose that this intensification of basal hypothalamic GABA release may underlie reported down regulation of corticosterone release in animals that underwent vagotomy procedure few days before measurement [11, 28]. As we report here, 30 days after vagotomy procedure, hypothalamic GABA concentration is even higher in inflammatory conditions. We think that this may be a result of enhanced external inhibitory signals, especially amygdalic GABA-GABA disinhibitory connections [42], which would inhibit activity of the inter-hypothalamic GABA circuits. Thus, the internal hypothalamic inhibition of CRF release would be suppressed, and in consequence, hypothalamic hormonal activity and further adrenal corticosterone release in response to inflammation, would be the same as in sham animals [22]. This is indeed what we observe in our studies. However, this hypothesis requires further, more detailed investigation.

We don’t know how the immune signals reach CNS in vagotomy conditions, at the early stages of inflammation. We think, that this may be an effect of an alternative mechanism replacing disrupted vagus-mediated signaling pathway. Wieczorek and Dunn [11] suggested, that this mechanism might be associated with changes in prostaglandin signaling. In fact, Dalli et al. [43] showed that even unilateral subdiaphragmatic truncal vagotomy, significantly alters macrophage lipid profile, which is involved in metabolism of arachidonic acid during intraperitoneal inflammation. Additionally, they reported that following left trunk vagotomy, the expression of COX2, an \(\)enzyme involved in PGE2 synthesis, significantly increased in macrophages [43]. We consider that elevation of inflammation-induced PGE2 synthesis may take over immune-to-CNS communication after vagotomy. We think that elevation of inflammation-induced PGE2 synthesis may take over immune-to-CNS communication after vagotomy, as we suggested previously [22]. In theory, blood-borne PGE2 reaches brain via bloodstream, where it acts upon epithelial cells of blood vessels. These vessels preserve intact prostaglandin-related cellular machinery after vagotomy [44]. Once at the brain, PGE2 activates CVOs, PVN and NTS [45, 46, 47, 48, 49] and, subsequently, other brain areas in a way similar to the now non-functional vagus nerve [49, 50].

Microarray and subsequent RealTime qPCR analysis suggest that transcriptome does not affect the activity of hypothalamus during inflammation in vagotomized rats after 30-days long recovery period. It would be interesting to see whether gene expression changes are more pronounced at earlier stages of recovery, while later giving way to other compensatory mechanisms such as neurotransmission alterations.

We found that amino acid neurotransmitters are deregulated in hypothalamus of vagotomized animals after prolonged recovery. This, along with our previous finding on deregulated monoamine systems in brain structures affecting functioning of hypothalamus suggests that vagal role in regulation of HPA axis after longer recovery period is replaced by changes in external neurotransmitter modulation of hypothalamic activity rather than alteration of basic hypothalamic functionality.

ACTH

adrenocorticotropic hormone

amygdala

ASP

aspartate

COX

cyclooxygenase

CNS

central nervous system

CRF

corticotropin-releasing hormone

CVOs

circumventricular organs

ELISA

enzyme-linked immunosorbent assay

EDTA

ethylenediaminetetraacetic acid

GABA

gamma-aminobutyric acid

GCs

glucocorticoids

GLU

glutamate

GLY

glycine

HIP

hippocampus

HPA

hypothalamic-pituitary-adrenal axis

HPT

hypothalamus

LPS

lipopolysaccharide

NTS

nucleus tractus solitarius

PAG

periaqueductal gray

PGE2

prostaglandin E2

PVN

paraventricular nucleus

RP-HPLC-ED

reversed phase high-performance liquid chromatography with electrochemical detection

RT-qPCR

quantitative real-time polymerase chain reaction

TNF-α

tumor necrosis factor alpha

TRP

tryptophan

TYR

tyrosine

Ethics approval and consent to participate

All applied experimental procedures were carried out with the approval of the Local Ethical Commission, 73/ŁB582/2012.

Consent for publication

Not applicable.

Availability of data and materials

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests

The authors declare that they have no competing interests

Funding

This work was supported by the Polish National Science Center (NCN, UMO 2012/07/B/NZ4/00205).

Authors' contributions

AK wrote the manuscript, isolated material for the microarray and qPCR analyses, performed the qPCR and HPLC analysis as well as statistical analysis of the HPLC and ELISA results.

AMS wrote the manuscript, oversaw and participated in isolation of the material for the microarray and qPCR analyses and coordinated them.

MW designed the experiment, performed the experimental procedures on living animals and collected the samples.

JG performed the bioinformatic and statistical analyses of microarray data.

MG, BB, and RIN performed the microarray analysis.

KK performed the ELISA tests.

Acknowledgements

Not applicable

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SupplementaryData.txt
SupplementaryTables.xlsx
Table S1. Samples used for microarray and qPCR experiments. Table S2. Primers. Table S3. Results of analysis of reference genes stability. Table S4. QPCR results.

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Hypothalamic neurochemical changes in long-term recovered bilateral subdiaphragmatic vagotomized rats

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Abstract

Figures

Background

Methods

Experiment

Samples preparation procedures

ELISA test for TNF-α

HPLC of amino acids

Microarray and RealTime qPCR

Microarray preparation:

qPCR analysis:

Results

TNF-α-based validation of inflammatory response (Figure 2.)

HPLC analysis (Figure 3.)

Microarray experiment (Figure 4.)

Discussion

Conclusion

Abbreviations

Declarations

References

Supplementary Files

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