Brainstem serotonergic, catecholaminergic, and inflammatory adaptations during chronic hypercapnia in goats

Abstract

Despite the prevalence of CO₂ retention in human disease, little is known about the adaptive neurobiological effects of chronic hypercapnia. We have recently shown 30-d exposure to increased inspired CO₂ (InCO₂) leads to a steady-state ventilation that exceeds the level predicted by the sustained acidosis and the acute CO₂/H⁺ chemoreflex, suggesting plasticity within respiratory control centers. Based on data showing brainstem changes in aminergic and inflammatory signaling during carotid body denervation–induced hypercapnia, we hypothesized chronic hypercapnia per se will lead to similar changes. We found that: 1) increased InCO₂ increased IL-1β in the medullary raphe (MR), ventral respiratory column, and cuneate nucleus after 24 h, but not after 30 d of hypercapnia; 2) the number of serotonergic and total neurons were reduced within the MR and ventrolateral medulla following 30 d of increased InCO₂; 3) markers of tryptophan metabolism were altered following 24 h, but not 30 d of InCO₂; and 4) there were few changes in brainstem amine levels following 24 h or 30 d of increased InCO₂. We conclude that these changes may contribute to initiating or maintaining respiratory neuroplasticity during chronic hypercapnia but alone do not account for ventilatory acclimatization to chronic increased InCO₂.—Burgraff, N. J., Neumueller, S. E., Buchholz, K. J., LeClaire, J., Hodges, M. R., Pan, L., Forster, H. V. Brainstem serotonergic, catecholaminergic, and inflammatory adaptations during chronic hypercapnia in goats.

Diseases such as chronic obstructive pulmonary disease (COPD) present a major health concern, with the incidence of death exponentially related to the degree of disease severity (1). A feature common to many respiratory-related diseases is the development of chronic hypercapnia and hypoxemia (2), which are secondary to pulmonary-airway limitations. Additionally, clinical situations requiring low-level ventilation strategies and multiple environmental conditions, such as aerospace and undersea exposure similarly result in the development of chronic hypercapnia, leading to morbidities such as increased risk of mortality, hypertension, and cognitive dysfunction. The isolated effects of hypoxemia within both respiratory disease and environmental exposure populations have been well studied (3, 4); however, the acclimatization to chronic hypercapnia, specifically the neurobiological effects of hypercapnia per se, have been limited in study.

We previously found that increasing the partial pressure of arterial carbon dioxide (PaCO₂ by 15 mmHg by inspired CO₂ (InCO₂) to 6% in goats resulted in a time-dependent hyperpnea that was not explained by measured levels of CO₂/H⁺ chemoreceptor stimuli (5). One previously proposed mechanism for such adaptation during exposure to chronic hypercapnia is through central plasticity of brainstem neuromodulatory systems, such as serotonin (5-HT) and catecholamines. For example, following carotid body denervation (CBD)-induced hypercapnia, the total number of neurons that express the rate-limiting enzyme for 5-HT synthesis [tryptophan hydroxylase (TPH)] are reduced (6), suggesting that chronic hypercapnia may result in the loss of central 5-HT. However, this decrease in the number of 5-HT neurons does not discern whether the loss is due to the reduced carotid afferents following CBD, or due to the hypercapnia experienced per se. Additionally, if indeed the loss of serotonergic neurons is due to the hypercapnia per se, the mechanisms resulting in the hypercapnic-induced serotonergic neuron loss remains to be determined.

One mechanism that may account for the loss of serotonergic neurons (or down-regulation of TPH) during chronic hypercapnia may be through increases in brainstem levels of inflammation. Accordingly, changes in circulating and CNS tissue levels of inflammation have been previously proposed to occur during acclimatization to chronic environmental stressors, such as hypercapnia and hypoxia. For example, chronic exposure to low levels of hypercapnia has been shown to increase the levels of circulating inflammatory microparticles (7, 8), and exposure to chronic hypoxia has been shown to increase mRNA expression of inflammatory markers (IL-1β, IL-6) within the nucleus tractus solitarius (NTS) (9). The secondary effects of this increase in inflammation are largely unknown; however, inhibition of inflammation through administration of the nonsteroidal anti-inflammatory drug (NSAID) ibuprofen attenuates ventilatory adaptations observed during chronic hypoxic exposure (9). The proposed changes in inflammation during altered environmental conditions, however, may have further neurologic implications, as increased levels of inflammatory molecules have been shown to lead to serotonergic neuron death in the dorsal raphe in vitro through an NMDA receptor-dependent excitotoxicity following altered tryptophan metabolism (10). Thus, although changes in inflammation may influence physiologic adaptations during chronically altered environmental conditions, these changes may similarly drive neurologic adaptations such as the loss of 5-HT neurons during hypercapnia. Accordingly, the objectives herein were to test the hypotheses that chronic hypercapnia leads to: 1) loss of serotonergic neurons of the brainstem medulla, 2) an increase in brainstem levels of inflammation, 3) changes in tryptophan metabolism within brainstem regions populated by serotonergic neurons, and 4) altered levels of 5-HT and other neuromodulators in brainstem cardiorespiratory control nuclei.

Ethical approval

All study protocols were reviewed and approved by the Medical College of Wisconsin Institutional Animal Care and Use Committee, which complies with the Public Health Services Policy on Humane Care and Use of Laboratory Animals and by extension all applicable provisions of the Animal Welfare Act and other federal statutes and regulations relating to animals. The Medical College of Wisconsin has remained continuously accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care, International since 1968 (000129). The investigators understand the ethical principles under which the journal operates, and the work herein complies with the journal animal ethics checklist.

Study population and conditions

Data were obtained from 30 female goats weighing 40–50 kg. In total, 18 of the 30 goats were studied for the physiologic changes during chronic hypercapnia; data from this cohort were included in a previous article (5). All goats were reared and transported under conditions specified by the U.S. Department of Agriculture. The goats were chronically housed and studied individually in 2 specially constructed plexiglass environmental chambers (3.5 × 4 × 6 ft); one chamber where the goat was maintained under normocapnic conditions and another in which the CO₂ levels could be increased. The temperature and relative humidity in the chambers were controlled and maintained within normal limits, and the photoperiods were fixed between 6 am and 6 pm daily. The goats were given access to feed and water ad libitum except during periods of study and being unfed for 24 h prior to surgery.

Experimental procedure

Two groups of goats were used for exposure to either 24 h or 30 d of room air or 6% elevated InCO₂. In total, 12 of the 30 goats were exposed to 24 h of room air (n = 6) or 6% InCO₂ (n = 6). Overall, 18 of the 30 goats were exposed to 30 d of room air (n = 9) or 6% InCO₂ (n = 9). Following 24 h exposure to 6% InCO₂ or room air, goats were euthanized, and brainstems were harvested for subsequent Western blotting and neurochemical (HPLC) analysis. No surgery or physiologic studies were completed on the goats in the 24-h group.

Goats exposed to 30 d of room air or 6% InCO₂ initially underwent surgical instrumentation and physiologic studies, both of which we previously reported in Burgraff et al. (5). Briefly, carotid arteries were elevated subcutaneously for serial blood sampling, electromyography (EMG) wires were implanted on the diaphragm, and a data logger was implanted subcutaneously for temperature and heart rate measurements. For physiologic studies, goats were allowed 2 wk of recovery following surgery while breathing room air. Following surgical recovery, baseline physiologic parameters were assessed, including inspiratory minute ventilation, breathing frequency, tidal volume, heart rate, blood pressure, arterial blood gasses, blood electrolytes, fractional concentration of expired oxygen (FeO₂) and CO₂ (FeCO₂), and the CO₂/H⁺ chemoreflex. After establishing baseline control values, goats were exposed to 30 d of either room air (21% O₂, 79% N₂, 0% CO₂) or 6% InCO₂ (19.5% O₂, 74.5% N₂, 6% CO₂). Physiologic studies were repeated regularly during exposure to either 30 d of room air or 6% InCO₂. The recorded physiologic parameters from goats exposed to 30 d of room air or 6% InCO₂ have been reproduced for reference from our previous publication (5) and are reported in Supplemental Tables S1 and S2. Following 30 d of exposure to 6% InCO₂ or room air, goats were euthanized, and brainstems were harvested for subsequent Western blotting, neurochemical (HPLC), or immunohistochemistry analysis. Due to differences in tissue preparation for immunohistochemistry and Western blotting, 6 of the 18 goats exposed to 30 d of room air or 6% InCO₂ (n = 3) were utilized for immunohistochemistry, whereas 12 of the 18 goats (n = 6) were utilized for Western blotting.

Tissue collection, immunohistochemistry, Western blotting, and HPLC analysis

Immunostaining protocol

Following euthanasia, the head was flushed (10% sucrose in PBS) and fixed (4% paraformaldehyde in PBS), and medullary and cortical tissue were extracted (within 6 min of euthanasia). Tissue was then cryoprotected in 20 and 30% sucrose for 72 h before freezing and serial sectioning (40 μm) in the transverse plane. Sections were floated in 6-well plates containing PBS prior to immunostaining. Control goat tissue and hypercapnic goat tissue were incubated simultaneously within the same solution utilizing a semipermeable membrane separating each well into 2 sides. Tissue slices were incubated in an antigen retrieval solution (Dako, Jena, Germany) at 80°C for 20 min, followed by a wash in PBS for 1 h. Tissue slices were then permeabilized with 0.4% Triton X-100/PBS for 10 min and blocked with 5% normal horse serum in 0.1% Triton X-100/PBS for 1 h. Following blocking, tissue slices were incubated with the primary antibody for either TPH (MilliporeSigma, Burlington, MA, USA) or neuronal nucleai (NeuN) (MilliporeSigma) (1:500 in 2.5% normal horse serum in 0.1% Triton X-100 PBS). After primary antibody incubation, tissue was washed in 0.1% Triton X-100/PBS for 3 × 10 min, followed by secondary antibody incubation for 30 min. Tissue was then incubated with an avidin-biotin complex (Vector Laboratories, Burlingame, CA, USA) for 30 min and washed for 3 × 10 min in PBS. Tissue was developed with 3,3′-diaminobenzidine (Dako) and subsequently mounted to electrostatically treated glass slides and cover slipped in hard-set mounting media. All antibodies were validated through the use of primary-omission controls showing lack of immunohistochemical staining.

Neuronal counts

The total number of TPH⁺ and NeuN⁺ neurons were counted within the medullary raphe (MR), ventrolateral medulla (VLM), cuneate nucleus (CN), and hypoglossal motor nucleus (HMN). For MR counts, 1 mm lateral to each side of the midline of the medulla was used extending from 0 to 12 mm rostral to obex. For VLM counts, 1 mm dorsal to the ventral surface of the medulla was used from 0 to 12 mm rostral to obex. For CN counts, a 1 mm² representative area was taken centrally from the CN extending 0–4 mm rostral to obex based on coordinates established previously (11). For HMN counts, all neurons within the HMN were counted between 0 and 3 mm rostral to obex based on coordinates established previously (11).

TPH and total neuronal (NeuN⁺) counts were performed manually in a blinded manner by an experienced laboratory technician. Neurons were denoted as positively stained for TPH based on a darkened cell soma with a noted nucleus void of immunohistochemical staining. Neurons were determined to be positively stained for NeuN based on both a darkened cell soma and nucleus. TPH neurons were counted within coronal slices taken every 40 μm and subsequently binned into 1 mm bins. NeuN neurons were counted within 40-μm-thick coronal slices taken every 1 mm. Representative images were taken with a Nikon E-400 (Nikon Instruments, Melville, NY, USA).

Western botting

Following euthanasia, brainstems were rapidly (<6 min following euthanasia) extracted and flash frozen with dry ice cooled methylbutane before storage for >1 d at −80°C. Stored brains and brainstems were sectioned into coronal slices (2 mm) at −20°C and tissue punches were obtained in homogenizing/loading buffer [tris-HCL (250 mM, pH 6.8), SDS, glycerol, βME, bromophenol, proteinase/phosphatase inhibitor] from nuclei of interest at 25 mg/ml (tissue w/v). Tissue punches were sonicated on ice. Criterion precast gels (10–20% Tris-HCl; Bio-Rad, Hercules, CA, USA) were prepared, wells washed, and SDS running buffer (10% Tris/glycol/SDS-tween in H₂O) was added to submerge entire gel. Five microliters of sample was added to each well, and electrophoresis was run at 200 V until dye front reached the bottom of the gel (∼30 min). Protein was transferred to PVDF membrane with Bio-Rad Trans-Blot Turbo Transfer for 3 min. Following transfer, membrane was incubated in blocking solution [2% bovine serum albumin (BSA) in 1xTBST] for 90 min and subsequently incubated in primary antibody solution for TPH (1:1000), NeuN (1:1000), IL-1β (1:500), indolamine dioxygenase (IDO) (1:500), quinolinic acid (QA) (1:500), 3-hydrokynurenine (3-OHK) (1:500), dopamine decarboxylase (DDC) (1:500), or ionized calcium binding adaptor molecule 1 (Iba1) (1:500) at 4°C on rocker overnight. Membrane was then rinsed 3 times with TBS-T, washed 3 times for 5 min each in TBS-T, and incubated in secondary antibody solution (1:10,000 HRP-2° in 2% BSA/1 time TBS-T). Blots were then rinsed 3 times with TBS-T, washed 3 times for 5 min each in TBS-T, and developed with Clarity Western ECL substrate (Bio-Rad) or femto developing solution (MilliporeSigma). For subsequent glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression analysis, blots were stripped with Restore Western blot stripping buffer for 5 min, followed by 3 times wash with TBS-T, washed 3 times for 5 min with TBS-T, blocked in blocking solution (2% BSA in 1xTBST) for 90 min, and reprobed with GAPDH primary antibody. Blots were imaged with a ChemiDoc imaging system (Bio-Rad) and processed with Image Lab software (Bio-Rad). Relative densities of proteins were always normalized to GAPDH to correct for protein loading.

HPLC analysis

Following euthanasia, the entire brain was rapidly (<6 min) extracted and flash frozen with dry ice cooled methylbutane before storage for >1 d at −80°C. Stored brainstems were sectioned into coronal slices (2 mm) at −20°C and tissue punches were obtained into 1.5 ml microcentrifuge tubes and stored at −80°C. For HPLC analysis, tissue punches were thawed in 0.1 M perchloric acid and wet weights determined before sonication and centrifugation at 10,000 rpm (20 min) at 4°C. Supernatant was removed from each sample and subjected to HPLC analysis for norepinephrine (NE), dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), 5-HT, 5-hydroxyindoleacetic acid (5-HIAA), and homovanillic acid (HVA). Standards with 3,4-dihydroxybenzylamine (DHBA) (internal standard) and 0.1 M perchloric acid were injected to establish values for quantification. Samples then underwent electrochemical detection [Bioanylitical Systems (BAS) LC4C; 0.65 V, 0.1nA, 0.1 Hz filter with Ag/Cl reference electrode] with a Waters µBondapak Column (3.9 × 300; Milford, MA, USA) at ambient temperature.

Data and statistical analysis

Differences between control and hypercapnic goat neuron counts were determined by a 2-way ANOVA with condition and distance as factors. A Holm-Sidak post hoc analysis was used to determine differences between conditions, with statistical significance determined as P < 0.05.

Western blots comparing TPH, NeuN, IL-1β, IDO, QA, 3-OHK, DDC, and Iba1 expression between hypercapnic goats and control room air goats were run across multiple blots as we have previously described (12). Western blots for each marker were run on individual blots such that there were 6 blots per nucleus. Additionally, 2 Western blots within each individual nucleus was necessary to complete the n = 6 analysis, with each Western blot consisting of n = 3. Expression of TPH, NeuN, IL-1β, IDO, QA, 3-OHK, DDC, and Iba1 were normalized to corresponding GAPDH expression to control for protein loading. Normalized expression values were averaged across control (n = 3) goats within each Western blot to determine mean control goat expression. GAPDH normalized expression of each band was then compared to mean control expression to derive expression relative to control. Data were presented as percent room air control expression. For TPH, NeuN, IL-1β, IDO, QA, 3-OHK, DDC, and Iba1, statistical differences between hypercapnic and room air control values were analyzed with a 1-way ANOVA, with statistical significance determined as P < 0.05.

HPLC results for brainstem tissue levels of 5-HT, catecholamines, and respective metabolites were analyzed through a 1-way ANOVA with condition as the factor. Holm-Sidak post hoc analysis was used to determine differences between conditions, with statistical significance determined as P < 0.05.

RESULTS

Total and serotonergic neuron counts in brainstem respiratory nuclei indicate unique effects of hypercapnia on the serotonergic neuronal populations

In goats exposed to chronic hypercapnia for 30 d, we found no differences from control goats in total neuron counts in either the CN or the HMN (Fig. 1). This finding was consistent across all rostral-caudal sections subjected to neuronal counting. In contrast, within the midline MR, there were ∼12% fewer NeuN⁺ neurons in goats exposed to 30 d of hypercapnia compared to 30 d room air controls (Fig. 2). The change in neurons was consistent across rostro-caudal distances within the nucleus. There were also ∼30% fewer NeuN⁺ neurons within the VLM in brainstems from goats exposed to 30 d of chronic hypercapnia (Fig. 2).

Figure 1.

No differences in the number of NeuN⁺ neurons were noted within the CN (A) and HMN (B) of goats exposed to 30 d of chronic hypercapnia compared to control goats exposed to 30 d of room air.

Figure 2.

The total number of NeuN⁺ neurons within the MR and VLM of goats exposed to 30 d of chronic hypercapnia were significantly lower than control goats exposed to 30 d of room air. A) Representative images showing a reduction in the number of NeuN⁺ neurons within the MR following 30 d of chronic hypercapnia. Images were taken at ×4 on a Nikon E-400 light microscope. B, C) NeuN⁺ neuron counts within the MR (B) and VLM (C) in goats exposed to 30 d of chronic hypercapnia and control goats exposed to 30 d of room air.

To determine if serotonergic neurons in these regions were among those lost, we also quantified TPH⁺ neurons. There were ∼40% fewer TPH⁺ neurons within the MR of goats exposed to 6% InCO₂ compared to goats exposed to room air (Fig. 3). The greatest difference in TPH⁺ neuron counts occurred within caudal sections extending from 0 to 6 mm rostral to obex, whereas rostral sections (6–12 mm rostral to obex) showed a smaller but still significant difference between room air and hypercapnic exposed goats (Fig. 3). Similarly, we found within the VLM region (1 mm dorsal to brainstem ventral surface) ∼30% fewer TPH⁺ neurons in hypercapnic vs. control goats. The greatest difference was within the caudal regions (0–4 mm rostral to obex), whereas less of a difference was noted in rostral regions extending 4–12 mm rostral to obex.

Figure 3.

The total number of TPH⁺ neurons within the MR and venterolateral medulla of goats exposed to 30 d of chronic hypercapnia were significantly lower than control goats exposed to 30 d of room air. A) Representative images showing a reduction in the number of TPH⁺ neurons within the MR following 30 d of chronic hypercapnia. Images were taken at 10× on a Nikon E-400 light microscope. B, C) TPH⁺ neuron counts within the MR (B) and VLM (C) in goats exposed to 30 d of chronic hypercapnia and control goats exposed to 30 d of room air.

Protein quantification confirms neuronal loss in the MR and VLM

Consistent with neuronal counts, there was 28% lower expression, as estimated by Western blot, of TPH within the caudal and rostral MR and 44% lower expression of NeuN in rostral MR from goats exposed to 30 d of hypercapnia compared to time-matched room air controls (Fig. 4 and Supplemental Fig. S2). Similarly, within the rostral VLM, there was 48% lower expression of TPH in the chronic hypercapnia goats relative to time controls, and 42% lower expression of NeuN within the rostral VLM in the goats exposed to 30 d of hypercapnia (Fig. 4 and Supplemental Fig. S2).

Figure 4.

A, B) Western blot expression of TPH showed a lower expression within both the rostral and caudal MR (A) and rostral VLM (B) following 30 d exposure to 6% InCO₂ (black bars) compared to time-matched controls exposed to 30 d of room air (gray bars). C, D) Additionally, Western blot expression of NeuN showed lower expression within the rostral MR (C) and VLM (D) following 30 d exposure to 6% InCO₂, compared to time-matched controls. *P < 0.05.

Transient changes in inflammation during chronic hypercapnia

Following 24 h exposure to 6% InCO₂, there was 39 and 40% greater expression of the inflammatory cytokine IL-1β within the caudal and rostral MR, respectively, compared to time controls (Fig. 5 and Supplemental Fig. S1). However, following 30 d exposure to 6% InCO₂, expression of IL-1β within the MR did not differ between hypercapnic and control goats (Fig. 7 and Supplemental Fig. S2). No changes in IL-1β expression were found following 24 h or 30 d exposure to 6% InCO₂ within the VLM compared to time controls (Figs. 6 and 8, and Supplemental Figs. S1 and S2).

Figure 5.

Following 24 h exposure to 6% InCO₂, Western blot expression within the caudal portions of the MR (0–2 mm rostral from obex) (A) revealed greater expression of IL-1β, IDO, TPH, and DDC compared to time-matched controls. Additionally, there was greater expression of IL-1β and IDO following 24 h exposure to 6% InCO₂ within the rostral portions of the MR (2–4 mm rostral from obex) (B), compared to time-matched controls. *P < 0.05.

Figure 7.

A) Following 30 d exposure to 6% InCO₂, Western blot expression within the caudal portions of the MR (0–2 mm rostral from obex) revealed lower expression of TPH compared to time-matched controls. B) Additionally, there was lower expression of IDO and TPH following 30 d exposure to 6% InCO₂ within the rostral portions of the MR (2–4 mm rostral from obex) compared to time-matched controls. *P < 0.05.

Figure 6.

A) Following 24 h exposure to 6% InCO₂, Western blot expression within the caudal portions of the VLM (0–2 mm rostral from obex) revealed greater expression of IDO compared to time-matched controls. B) Additionally, there were no changes noted within the rostral portions of the VLM (2–4 mm rostral from obex) following 24 h exposure to 6% InCO₂ compared to time-matched controls. *P < 0.05.

Figure 8.

Following 30 d exposure to 6% InCO₂, A) Western blot expression within the caudal portions of the VLM (0–2 mm rostral from obex) showed no differences in any measured marker of inflammation or tryptophan metabolism compared to time-matched controls. B) Additionally, there was lower expression of IDO, QA, TPH, and DDC following 30 d exposure to 6% InCO₂ within the rostral portions of the VLM (2–4 mm rostral from obex) compared to time-matched controls. *P < 0.05.

Across other nuclei investigated, there was significantly greater expression of IL-1 β following 24 h exposure to 6% InCO₂ within ventral respiratory column (VRC) and CN compared to time controls (Fig. 9). In contrast, there were no differences in IL-1β expression in the 24 h exposure groups across all other nuclei tested, including the solitary complex (dorsal motor nucleus of the vagus and NTS), VLM, retrotrapezoid nucleus (RTN), and the HMN (Table 1). Within the regions in which IL-1β expression was increased after 24 h of hypercapnia, levels of IL-1β returned to control levels by 30 d of hypercapnia. Additionally, no differences in IL-1β expression between goats exposed to 30 d of hypercapnia or room air were found in other nuclei investigated (solitary complex, VLM, RTN, HMN, and CN) (Table 1).

Figure 9.

Western blot expression of IL-1β showed increased protein expression within the MR, VRC, and CN in goats exposed to 24 h of chronic hypercapnia compared to control goats exposed to 24 h of room air.

TABLE 1.

GAPDH normalized IL-1β Western blot expression relative to control following 24 h or 30 d exposure to 6% InCO₂

Variable	24 h (%)	30 d (%)
Solitary complex	115 ± 10	118 ± 15
VLM	97 ± 11	87 ± 11
RTN	103 ± 11	106 ± 10
MR	139 ± 14*	92 ± 11
VRC	144 ± 8*	96 ± 10
Hypoglossal	109 ± 12	111 ± 14
Cuneate	151 ± 5*	107 ± 11

Following 24 h exposure to 6% InCO₂, there were significant increases in IL-1β protein expression within the MR, VRC, and CN compared to control goats exposed to 24 h of room air. *P < 0.05. No other differences in IL-1β protein expression were noted following 24 h or 30 d exposure to 6% InCO₂ compared to control goats exposed to room air across all other nuclei investigated.

Changes in Iba1 expression following 30 d of chronic hypercapnia

To examine whether microglial activation occurred during chronic hypercapnia, Iba1 expression was determined following exposure to either 24 h or 30 d of room air or 6% elevated InCO₂. Following 24 h exposure to 6% InCO₂, there were no differences in Iba1 expression within the rostral or caudal MR or VLM compared to time controls. However, following 30 d exposure to 6% InCO₂, there was 40% greater expression of Iba1 within the caudal MR, and 49% lower expression of Iba1 within the rostral VLM, compared to time controls (Fig. 10 and Supplemental Fig. S3).

Figure 10.

Western blot expression of Iba1 showed no difference in protein expression between goats exposed to 24 h of 6% InCO₂ compared to time-matched control. However, following 30 d exposure to 6% InCO₂, Iba1 expression was significantly greater within the caudal raphe and rostral VLM compared to goats exposed to 30 d of room air. *P < 0.05.

Changes in tryptophan metabolism during chronic hypercapnia

To test whether changes occurred in tryptophan metabolism within the MR and VLM during chronic hypercapnia, we measured expression of the major enzymes and protein levels along the TPH and IDO-dependent pathways of tryptophan metabolism. Within the MR, following 24 h exposure to 6% InCO₂, compared to control goats, there was 39 and 35% greater expression of the enzyme IDO within the caudal and rostral portions of the MR, respectively (Fig. 5 and Supplemental Fig. S1). An increase in the enzyme, IDO, may enhance tryptophan breakdown to either QA or 3-OHK, but these downstream substances did not differ between control and hypercapnic goats within the rostral or caudal MR (Fig. 5 and Supplemental Fig. S1). Additionally, there was 34% greater expression of TPH and 48% greater expression of DDC within the caudal portion of the MR of goats exposed to 24 h of 6% InCO₂, compared to control, with no changes in TPH or DDC noted in rostral portion of the MR (Fig. 5 and Supplemental Fig. S1).

Within the VLM, IDO showed 46% greater expression within the caudal portions following 24 h of hypercapnic exposure (Fig. 6 and Supplemental Fig. S1). However, similar to the MR, there were no subsequent changes in either QA or 3-OHK within the VLM following 24 h hypercapnic exposure. No changes were found in either TPH or DDC within the VLM following 24 h exposure to 6% InCO₂, compared to control (Fig. 6 and Supplemental Fig. S1).

Any changes in IDO found following 24 h hypercapnic exposure were not found following 30 d hypercapnic exposure (Figs. 7 and 8 and Supplemental Fig. S2). Additionally, there were no differences in expression of QA or 3-OHK following 30 d exposure to 6% InCO₂, compared to control, with the exception of a 28% decrease in expression of QA within the rostral portion of the VLM (Fig. 8 and Supplemental Fig. S2).

TPH expression was found to be reduced by 28% within both the caudal and rostral portions of the MR following 30 d exposure to 6% InCO₂, with no changes in DDC, compared to control (Fig. 7 and Supplemental Fig. S2). Similarly, there was a 48% decrease in TPH expression, and 49% lower expression of DDC within the rostral portion of the VLM following 30 d exposure to 6% InCO₂, with no change in TPH or DDC expression in the caudal portion of the VLM, compared to control (Fig. 8 and Supplemental Fig. S2).

Brainstem amine levels were largely unchanged after 24 h or 30 d of chronic hypercapnia

Given the greater than predicted steady-state ventilation in goats exposed to chronic hypercapnia over 30 d (5), we tested the hypothesis that excitatory neuromodulators would be increased within several nodes of the neural network controlling breathing. We found following 30 d of exposure to 6% InCO₂, there was a significantly greater concentration of the metabolic breakdown product of DA, HVA within the NTS (Fig. 11), along with a significant decrease in DOPAC (Fig. 12) compared to goats exposed to 30 d of room air. Additionally, there was a significant decrease in HVA within the VRC, and a significant decrease in NE within the VLM in goats exposed to 30 d of 6% InCO₂, compared to time controls (Fig. 11). These differences were not evident in goats exposed to 24 h of 6% InCO₂, compared to goats exposed to 24 h of room air. There were no other differences in tissue levels of catecholamines or catecholamine metabolites (NE, DA, DOPAC, HVA), 5-HT, or the metabolic breakdown product of 5-HT, 5-HIAA between goats exposed to either 24 h of 6% InCO₂ or room air, or goats exposed to 30 d of 6% InCO₂ or room air (Figs. 11–13).

Figure 11.

HPLC analysis of NE and HVA shows a significant (P < 0.05) decrease in NE within the VLM, increase in HVA within the NTS, and decrease in HVA within the VRC of goats exposed to 30 d of chronic hypercapnia compared to goats exposed to 30 d of room air. No other differences in HVA or NE were found within the NTS, VRC, RTN, MR, and VLM of goats exposed to 24 h or 30 d of chronic hypercapnia compared to goats exposed to 24 h or 30 d of room air. *P < 0.05.

Figure 12.

HPLC analysis of DA and DOPAC shows a significant decrease in DOPAC within the NTS of goats exposed to 30 d of chronic hypercapnia compared to goats exposed to 30 d of room air. No other differences in DA or DOPAC were found within the NTS, VRC, RTN, MR, and VLM of goats exposed to 24 h or 30 d of chronic hypercapnia compared to goats exposed to 24 h or 30 d of room air. *P < 0.05.

Figure 13.

HPLC analysis of 5-HT and 5-HIAA showed no differences in 5-HT and 5-HIAA within the NTS, VRC, RTN, MR, and VLM of goats exposed to 24 h or 30 d of chronic hypercapnia compared to goats exposed to 24 h or 30 d of room air.

Within the RTN and MR, there were no significant differences in measured levels of all catecholamines (NE, DA) or metabolic breakdown products (DOPAC, HVA), 5-HT, or the metabolic breakdown product of 5-HT (5-HIAA) in goats exposed to either 24 h or 30 d of 6% InCO₂ or room air (Figs. 11–13).

DISCUSSION

The incidence of death in patients retaining CO₂ with diseases such as COPD is greater than those with similar disease states that maintain levels of PaCO₂ (1, 13, 14), suggesting that chronic hypercapnia per se may have negative consequences on long-term prognosis and may independently contribute to higher mortality. Additionally, volitional implementation of hypercapnia within patients requiring mechanical ventilation to prevent barotrauma has been shown to be associated with an increased incidence of death through secondary effects on muscle and alveolar function (15). The CNS adaptations that occur during conditions of chronic CO₂ retention are largely unknown, but may be governed by known or novel mechanisms of neuroplasticity that could present important molecular targets for disease management, and preventing disease progression and risk of mortality. We previously reported the ventilatory and integrated physiologic adaptations that occur during chronic exposure to 6% elevated InCO₂ in adult goats (5). The predominant finding was the development of physiologic adaptations that could not be explained by chemical stimuli alone, suggesting the presence of hypercapnic-induced neuroplasticity. To gain insight into central mechanisms of plasticity that may occur during chronic hypercapnia, we tested the hypothesis that chronic hypercapnia leads to: 1) loss of serotonergic neurons of the brainstem medulla, 2) an increase in brainstem levels of inflammation, 3) changes in tryptophan metabolism within serotonergic neuron populations of the brainstem medulla, and 4) altered levels of 5-HT and other excitatory neuromodulators within brainstem cardiorespiratory control nuclei. Our findings partially support our hypothesis that chronic hypercapnia leads to: 1) a loss of serotonergic neurons through a reduction in the number of both TPH⁺ and NeuN⁺ neurons within sites of 5-HT production (MR and VLM), 2) increases in inflammation through a transient site-specific increase in IL-1β expression, and 3) altered tryptophan metabolism through changes in key regulatory enzymes in the breakdown of 5-HT. However, our findings do not support the hypothesis that levels of 5-HT or other excitatory neuromodulators are altered during chronic hypercapnia.

Chronic hypercapnia decreases the number of brainstem 5-HT neurons

The primary sites of serotonergic neuron somata, which express TPH, are located within the midline MR nuclei and the parapyramidal regions of the VLM (16). TPH-expressing neurons within these 2 regions are postulated to exert effects on ventilation and other physiologic mechanisms through 2 major mechanisms: 1) a tonic, excitatory input to the respiratory control network and 2) their intrinsic sensitivity to changes in CO₂ and pH (17–19). Thus, increased levels of CO₂/H⁺ likely cause increased activity of at least a subset of brainstem TPH⁺ neurons, subsequently leading to an increase in excitatory neuromodulator (5-HT) release to target nuclei including the VRC, NTS, and RTN/parafacial respiratory group (pFRG) (20–22). Adaptations in 5-HT signaling during exposure to chronic hypercapnia have been limited in study; however, reductions in the number of 5-HT (TPH⁺) neurons of the brainstem medulla following CBD-induced hypercapnia (6) suggest chronic hypercapnia alone may result in CNS adaptations through changes in the 5-HT system. An important consideration, however, is that CBD causes hypoventilation-induced chronic hypercapnia (6, 23), and the reduction in TPH⁺ neurons in the CBD model could have resulted from the elimination of tonic afferent signaling from the peripheral chemoreceptors to the neural respiratory network or the chronic hypercapnia per se. The data presented herein, similar to CBD-induced hypercapnia, show that chronically elevated environmental CO₂-induced hypercapnia also causes a reduction in the number of TPH⁺ neurons within the raphe nuclei, as well as in the VLM, suggesting that chronic hypercapnia per se can lead to deficiencies in the 5-HT system. However, a reduction in the number of TPH⁺ immunoreactive neurons following chronic hypercapnia does not discern whether the reduction is due to a reduction in TPH expression within serotonergic neurons or a loss of serotonergic neurons. The data showing a reduced number of total (NeuN⁺) neurons in these regions after 30 d of hypercapnia do however strongly suggest that the reduced TPH⁺ neuronal counts may indeed be due to serotonergic neuron loss rather than a reduced enzyme expression. Moreover, it appears that the raphe and VLM nuclei were rather selectively affected because we found no differences in total neuron counts within the nonrespiratory CN and respiratory-related HMN after 30 d of chronic hypercapnia. Although we did not attempt an exhaustive cell count of all respiratory nuclei, the data provide evidence that a major consequence of chronic hypercapnia per se may be a relatively selective neuronal loss in the raphe and VLM nuclei and specifically loss of serotonergic neurons.

Alterations in inflammation during chronic hypercapnia

Chronic challenges to the respiratory control system (hypoxia) have been shown to elicit inflammatory signaling responses, which may also be altered in conditions of chronic hypercapnia (9). Thus, we tested whether changes in brainstem levels of inflammation (IL-1β) occurred during 24 h or 30 d of 6% InCO₂ compared to 24 h or 30 d exposure to room air. Following 24 h of 6% InCO₂, IL-1β protein expression was elevated within 3 nuclei of the brainstem medulla including the MR, VRC, and CN. The increase in IL-1β showed a temporal pattern because this increase in IL-1β was not present following 30 d exposure to 6% InCO₂. Although the functional consequences of these changes in inflammatory signaling remain to be tested, similar observations have been made in other studies providing evidence of a functional contribution to ventilatory acclimatization to chronic hypoxia. Thus, increases in inflammation during exposure to chronic hypercapnia may contribute to the ventilatory acclimatization that occurs during hypercapnic exposure through inflammatory cytokine modulation of neuronal excitability (24). Indeed, if this is the case during chronic hypercapnia, therapeutic modulation of inflammation through anti-inflammatory therapy may uncover specific molecular mechanisms involved in the CNS adaptations occurring during chronic CO₂ retention. It may also imply that we should do more to keep PaCO₂ levels as low as we can in COPD patients with hypercapnia.

Changes in tryptophan metabolism during chronic hypercapnia

CNS tissue levels of inflammation during chronic hypercapnia may also represent a mechanism leading to the loss of serotonergic neurons during chronic hypercapnia. The data presented herein, along with evidence following CBD strongly suggest that chronic hypercapnia results in a loss of serotonergic neurons of the brainstem medulla; however, the mechanism leading to the presumptive loss in 5-HT neurons is unknown. Under normal conditions, tryptophan may be broken down through 2 discreet pathways. In 1 case, tryptophan may be converted to 5-HT through a TPH-dependent pathway, whereas the alternative is through an inflammatory-sensitive IDO-dependent pathway, whereby the breakdown of tryptophan results in the formation of the neurotoxic substances QA and 3-OHK. Previous studies in vitro of serotonergic neurons in the dorsal raphe have shown that increasing markers of inflammation leads to an increase in IDO expression, resulting in neuronal death through a presumed increase in the neurotoxins QA and 3-OHK (10). Thus, if alterations in levels of inflammation and tryptophan metabolism occur during exposure to chronic hypercapnia, this may represent a mechanism resulting in a loss of serotonergic neurons. Following 24 h exposure to hypercapnia, there was an increase in IL-1β expression within the MR, suggesting an increase in inflammation following initial exposure to 6% elevated InCO₂. We also found an increase in the inflammatory-sensitive enzyme, IDO, suggesting that 24 h of hypercapnia may result in a “shift” in tryptophan metabolism toward the IDO-dependent pathway within the MR, resulting in neuronal death by increases in neurotoxic substances following 30 d of chronic hypercapnia. However, following 24 h of hypercapnia we did not find measurable changes in either QA or 3-OHK.

Within the VLM, we did not find any changes in IL-1β expression following 24 h exposure to hypercapnia. However, despite the lack of change in this inflammatory marker, we found there to be a regionally specific increase in IDO, suggesting that only portions of the VLM may result in a change in tryptophan metabolism during chronic hypercapnia. Additionally, the lack of change in IL-1β within the VLM suggests that the increase in IDO expression was independent of IL-1β expression and may be due to increases in other unmeasured inflammatory molecules.

Following 30 d exposure to chronic hypercapnia, expression of IL-1β returned to control levels within the MR and remained unchanged in the VLM. This occurred along with expression of IDO that was found to be at or below control levels. These data are consistent with a time-dependent nature of an inflammatory response during chronic hypercapnia. This time dependency of inflammation was similarly found across other brainstem nuclei during chronic hypercapnia within the VRC and CN. Accordingly, transient changes in inflammation during chronic hypercapnia are consistent with a hypothesis previously proposed (12), whereby exposure to chronic hypercapnia results in an initial induction neuroplasticity phase, followed by a remodeling phase where initial neuroplastic changes revert to control levels in order to maintain a new steady state during the chronically elevated chemical stimuli.

Microglial activation state during chronic hypercapnia

To determine whether the transient increase in IL-1β expression was associated with microglial activation, we measured tissue levels of Iba1, indicative of microglial activation. Interestingly, we found no changes in Iba1 expression following 24 h exposure to 6% InCO₂, indicating that the initial increase in IL-1β expression was likely not due to microglial activation. However, following 30 d exposure to 6% InCO₂ we found site-specific changes in Iba1 expression, suggesting delayed alterations in local immune responses during chronic hypercapnia. Following 30 d exposure to 6% elevated InCO₂, Iba1 expression was elevated within the MR, suggesting a heightened immune response within the MR during the chronic phase of hypercapnic exposure. This finding, however, contrasted with a decreased expression in Iba1 within the VLM following 30 d of chronic hypercapnia, which may indicate a local immune deficiency within the VLM during the chronic phase of hypercapnic exposure. The functional consequences of locally altered microglial activation following 30 d of hypercapnia remain unknown; however, further studies investigating the balance between the pro- and anti-inflammasomes and their interaction with immune modulation may provide valuable insight into the progression of neuropathology and adaptation during chronic hypercapnia.

Levels of tissue 5-HT during chronic hypercapnia

Based on the loss of serotonergic neurons following CBD-induced hypercapnia, and the loss of serotonergic neurons shown here, we hypothesized that levels of 5-HT throughout brainstem nuclei would be subsequently reduced during chronic exposure to 6% InCO₂. Remarkably, despite the reduction in serotonergic neuron numbers within the MR and VLM following 30 d of chronic hypercapnia, we found no changes in tissue levels of 5-HT within the serotonergic neuron populations of the MR and VLM. Similarly, no changes in 5-HT were found within serotonergic neuron target fields in the NTS, VRC, and RTN, whom receive direct serotonergic input from either or both the MR and VLM (20–22). The finding of reduced TPH neuron expression throughout the brainstem medulla with no subsequent decrease in 5-HT within direct synaptic targets likely reflects that under room air conditions TPH activity within the brainstem functions below enzymatic V_max. Thus, a reduction in TPH⁺ neuron expression may reduce the quantity of available enzyme for 5-HT production but may be fully compensated through an increase in TPH activity within remaining serotonergic neurons. Alternatively, 5-HT levels may be normal under steady-state conditions during chronic hypercapnia but may be deficient during other dynamic challenges, which were not tested here. The physiologic consequences of the presumptive loss of hindbrain serotonergic neurons during chronic hypercapnia were beyond the scope of this study; however, it may be postulated that the reduction in neurons may be a compensatory response in order to prevent overexcitation within the respiratory control network during chronic increased InCO₂ by maintaining normal levels of 5-HT throughout the brainstem, concurrent with a chronically elevated stimulus.

Catecholamine signaling during chronic hypercapnia

Lastly, to test whether changes occur in brainstem levels of excitatory neuromodulators during chronic hypercapnia, we investigated whether tissue catecholamines, and their metabolites were altered during chronic exposure to 6% elevated InCO₂. Exposure to acute hypercapnia (minutes) can increase circulating catecholamines within the blood (25); however, this effect of acute hypercapnia is not the same within CNS tissue levels of catecholamines. Catecholaminergic neurons within the CNS are located within 14 cell groups denoted A1–A14, in which A1–A7 contain NE-producing neurons (26, 27) and dopaminergic neurons reside within groups A8–A14 (27). Like 5-HT neurons, subsets of these neuronal populations have been shown to be sensitive to changes in CO₂/pH (28, 29), suggesting that the activity of some catecholaminergic neurons are likely increased during chronic hypercapnia. Consistent with this idea are data showing that acute exposure to environmental hypercapnia (30 min–24 h) results in time-dependent alterations in the concentrations of NE and DA throughout the CNS through changes in neuromodulator production and utilization (30–32). The effects of longer exposures (>24 h) to elevated InCO₂ on brainstem levels of catecholamines, however, have not been previously studied. With a few exceptions, we observed no changes in catecholamines and their metabolic breakdown products in the brainstem nuclei tested after 24 h or 30 d of chronic hypercapnia. The only significant changes in brainstem tissue levels of catecholamines noted were an increase and decrease in the concentration of HVA and DOPAC, respectively, within the NTS, a decrease in NE within the VLM, and a decrease in HVA within the VRC following 30 d of chronic hypercapnia. The increase in HVA and decrease in DOPAC may be indicative of an increase in the activity of catechol-O-methyltransferase subsequent to an increase in DA metabolism within the NTS following 30 d of exposure to 6% elevated InCO₂. Despite the decrease in DOPAC and increase in HVA, we found no change in NTS levels of DA, suggesting that DA production and subsequent release within the NTS may have increased in proportion to the increase in DA metabolism in order to maintain constant levels during chronic hypercapnia. The decrease in HVA within the VRC, however, may represent a decrease in DA metabolism in the VRC secondary to a decrease in DA release, evident by no change in DA levels. Within the VLM, the mechanisms resulting in a decrease in NE remain unknown; however, this may represent a loss of NE stores within the chemosensitive catecholaminergic neuron population of VLM following chronic chemical stimulation or conversely may represent a compensatory mechanism to reduce excitatory neuromodulator release during chronic excitation. Regardless, there were no other changes in tissue catecholamines following 24 h or 30 d of chronic hypercapnia, suggesting that changes in catecholaminergic signaling likely do not occur during chronic (>24 h) hypercapnia within our model.

It is noteworthy that there was a relatively large between-goat variation in the tissue levels of 5-HT and catecholamines within control goats measured in our study. However, exposure to chronically elevated InCO₂ resulted in greater variation among individual goats in brainstem tissue levels of catecholamines and 5-HT. For example, following 30 d of chronic hypercapnia, there was significantly greater (F > F_Crit) variation among individual goat data for tissue levels of DA, DOPAC, 5-HIAA, and 5-HT compared to goats exposed to 30 d of room air. Importantly, this variation occurred despite the consistency of physiologic adaptations across goats studied, as previously reported (5). Large variations among tissue catecholamines and 5-HT was similarly shown following CBD-induced hypercapnia (6) despite consistent physiologic adaptations, highlighting the individualized response in tissue catecholamines and 5-HT among goats during times of chronic CO₂ retention. Thus, the findings of increased variation among catecholamine and 5-HT responses to chronic hypercapnia may highlight unique, and possibly genetically determined neurochemical adaptive mechanisms across goats that ultimately result in similar physiologic responses. Additionally, variation among measured catecholamines following chronic hypercapnia may represent differences in gene expression responses (33) within individual goats used herein for study.

Importance of present findings

The data herein provide evidence that chronic hypercapnia per se leads to: 1) a relatively selective reduction in brainstem serotonergic neurons, 2) site- and time-dependent increases in a brainstem inflammation following 30 d of chronic hypercapnia, 3) time-dependent alterations in tryptophan metabolism that may preferentially “shift” the breakdown of tryptophan to the IDO pathway, 4) a delayed microglial response that may indicate immune cell activation or suppression throughout discreet brainstem nuclei, and 5) minimal alterations in excitatory neuromodulators, including 5-HT and catecholamines. Although the mechanistic significance of these findings remains to be established, it appears that the reduction in hindbrain serotonergic neurons is likely due to chronic hypercapnia per se—a finding that may be present in humans suffering from respiratory-related diseases resulting in chronic CO₂ retention and patients exposed to permissive hypercapnia through mechanical ventilation. The cause for the loss in serotonergic neurons is unknown; however, the data presented herein suggest that increased breakdown of tryptophan through the IDO pathway may be a likely mechanism within the MR. Further, we speculate that the loss of serotonergic neurons may represent a compensatory adaptation to chronic hypercapnia, which could represent a major challenge in managing patients that retain CO₂ as normalizing PaCO₂ may remove a stimulus to breathe in an adapted control system. It may also explain difficulties in removing patients from mechanical ventilation following permissive hypercapnia, depending upon the duration and magnitude of the hypercapnia.

Our data also point to the presence of brainstem inflammation during chronic hypercapnia. Further studies investigating this phenomenon may provide critical information for practitioners managing patients with hypercapnia by determining a functional role of hypercapnia-induced inflammation in the brainstem and its potential link to 5-HT neuron loss. Finally, although there were measured changes in brainstem neuromodulatory systems, there is a lack of a clear correlation between any measured changes in neuromodulatory systems and physiologic adaptation to chronic hypercapnia. Thus, future study through intervention of the specific adaptations presented herein are needed to fully address the hypothesis that changes in brainstem neuromodulators contribute to physiologic adaptation to chronic hypercapnia.

Glossary

3-OHK

3-hydroxykynurenine

5-HIAA

5-hydroxyindoleacetic acid

5-HT

serotonin

BSA

bovine serum albumin

CBD

carotid body denervation

CN

cuneate nucleus

COPD

chronic obstructive pulmonary disease

DA

dopamine

DDC

dopamine decarboxylase

DOPAC

3,4-dihydroxyphenylacetic acid

GAPDH

glyceraldehyde 3-phosphate dehydrogenase

HMN

hypoglossal motor nucleus

HVA

homovanillic acid

Iba1

ionized calcium binding adaptor molecule 1

IDO

indolamine dioxygenase

InCO₂

inspired CO₂

MR

medullary raphe

NE

norepinephrine

NeuN

neuronal nuclei

NTS

nucleus tractus solitarius

PaCO₂

partial pressure of carbon dioxide

QA

quinolinic acid

RTN

retrotrapezoid nucleus

TPH

tryptophan hydroxylase

VLM

ventrolateral medulla

VRC

ventral respiratory column

AUTHOR CONTRIBUTIONS

N. J. Burgraff performed surgeries and experiments, analyzed data, created figures, and wrote the manuscript; S. E. Neumueller performed experiments and manuscript editing; K. J. Buchholz performed experiments; J. LeClaire performed experiments; M. R. Hodges performed surgeries, contributed to intellectual discussions, and manuscript editing; L. Pan performed surgeries and contributed to intellectual discussions; and H. V. Forster contributed to intellectual discussions and manuscript writing and editing.

Supplementary Material

This article includes supplemental data. Please visit http://www.fasebj.org to obtain this information.