Neuroinflammation contributes to autophagy flux blockage in the neurons of rostral ventrolateral medulla in stress-induced hypertension rats

Seventy-two 8-week-old male Sprague-Dawley rats weighting 250–300 g were obtained from the animal center of Fudan University. They were housed at a 12 h light/dark cycle with free access to food and water; room temperature was maintained between 23 and 24 °C. All experimental procedures conformed to both Fudan University and international guidelines on the ethical use of animals; all efforts were made to minimize the number of animals used and their suffering. The rats were divided into three groups (n = 24): a normotensive rats group (control), SIH group, and SIH plus minocycline application (Mino + SIH) group. The SIH model was established as previously described [2‐4]. Briefly, rats in the stressed groups were placed in a cage (22 cm × 22 cm × 28 cm) with a grid floor and subjected to electric foot shocks. The delivery of intermittent electric shocks (35–75 V, 0.5 ms in duration) through the grid floor every 2–30 s was randomly controlled by a computer. Noises (range, 88–98 dB) produced by a buzzer were given synchronously as a conditioned stimulus. The computer program was designed to give two types of stimuli: noises plus electric foot shocks or noises only, both of which were delivered randomly. On day 7 when SBP and heart rate (HR) of the stressed groups increased to a nearly stable level, the computer linked to the stressor apparatus was adjusted, gradually decreasing electric foot-shock times and prolonging the interval between shocks until only noise stimuli remained. The control group of rats underwent sham stress; they were put into the cage for the same period of time, but were neither subjected to foot shocks nor noises. Rats were subjected to stress for 2 h twice daily for l4 consecutive days. Two hours after being subjected to stress, SBP was consciously measured using the tail-cuffed method. Measurements were repeated three times and the average value was taken as the SBP (Additional file 1: Figure S1).

Before the rats were sacrificed, blood pressure was measured via a femoral artery cannula using a pressure transducer and a polygraph (Model SMUP-A, Department of Physiology and Pathophysiology, Shanghai Medical College of Fudan University, Shanghai, China). The HR was derived automatically from the arterial blood pressure phasic wave by the computer. During the experiment, body temperature was measured with a rectal thermometer and maintained at 37.5 ± 0.5 °C by a temperature controller (H-KWDY-III, Quanshui Experimental Instrument, Chengdu, China). Arterial blood PaO₂ and PaCO₂ were periodically monitored using a blood gas analyzer (EasyBloodGas, Medica Corporation, Bedford, MA, USA) and maintained within normal limits. Arterial blood pH was maintained between 7.35 and 7.45.

Power spectral analysis of SBP signals was performed in animals with sodium pentobarbital (50 mg kg⁻¹, i.p.) anesthesia. SBP signal was monitored for 30 min by femoral artery cannulation. Power spectral analysis of SBP signals was performed by fast Fourier transform. The low frequency (LF, 0.25–0.8 Hz) component of the SBP spectrum was taken as it was originally from the RVLM [26, 27], and reflected the prevalence of sympathetic nerve activity to the vessels.

The procedures were performed as similar study [15]. Briefly, the animals were anesthetized with pentobarbital sodium (50 mg kg⁻¹, i.p.), then the osmotic minipump was implanted into the cisterna magna for infusion of minocycline. A midline dorsal neck incision was made, and the dura mater between the foramen magnum and C1 lamina was exposed following dissection of muscles. The dura was perforated with a 22-gauge steel needle. After observation of cerebrospinal fluid leakage from this hole, a PE-5 catheter (Clay Adams, Sparks, MD) was advanced for 5 mm into the cisterna magna. The catheter was sealed to the dura with tissue glue and incision was closed with layered sutures. The outer end of the catheter was connected to a micro-osmotic minipump (Alzet 1007D, DURECT Co., Cupertino, CA), which was placed under the skin in the neck region. The osmotic minipump (implanted SC) was connected to the infusion cannula via the catheter tube delivering agent. Inhibitor of microglial activation minocycline (Mino, 0.3 μl/h, Sigma-Aldrich, St. Louis, MO) was delivered by osmotic minipump for 1 week on the basis of published in vivo [15, 28, 29]. Animals received procaine penicillin (1000 IU, IM) injection postoperatively, and only animals that showed progressive weight gain after the operation were used in subsequent experiments. Control infusion of artificial cerebrospinal fluid (aCSF) was served as volume and vehicle control. Minocycline solution was prepared by dissolving 172 ng minocycline in 1 mL normal aCSF.

In the process of autophagosome synthesis, the cytoplasmic localization of microtubule-associated protein 1 light chain 3-I (LC3-I) is conjugated with phosphatidylethanolamine and becomes the membrane-bound form LC3-II, which is located in both the outer and inner membranes of the autophagosomes. Due to this property, the most widely used assay for monitoring autophagosomes is the observation and quantification of fluorescent-tagged LC3 (such as GFP-LC3, RFP-LC3) puncta LC3-II by fluorescence microscopy [20]. Monitoring autophagic flux is important for the analysis of autophagy. Tandem fluorescent-tagged LC3 (tf-LC3) is a convenient assay for monitoring autophagic flux based on different pH stability of monomeric red fluorescence protein-enhanced green fluorescence protein-LC3 (mRFP-EGFP-LC3) proteins [20]^.

The gene transfer of tf-LC3 and/or GFP-LC3 into the RVLM was performed by microinjection bilaterally into the nucleus of adeno-associated virus (AAV) RFP-GFP-tandem fluorescent LC3/GFP-LC3 14 days prior to stress application. An adenoviral suspension containing 5 × 10⁸ plaque-forming units (pfu)/100 nl mediated gene transferring into the RVLM was administered into each injection site over 10–15 min by pressure through a glass micropipette.

Intra-RVLM microinjection and hemodynamic measurements were performed as described previously [2‐4]. Briefly, the rats were anesthetized with a mixture of urethane and chloralose comprised of 140 g urethane, 7 g chloralose, and 7 g borax per 1 l normal saline in an intraperitoneal dose of 7 ml/kg. Tracheas were intubated with polyethylene tubes, and the animals spontaneously breathed room air. Heads of the rats were affixed on a stereotaxic apparatus (Neurostar Tubingen, Germany) and flexed to an angle of approximately 45°. The occipital bone was carefully removed to expose the fourth ventricle, the floor of which was kept horizontally. A glass micropipette was inserted into the RVLM (1.5–1.9 mm ahead of the obex, 1.5–2.0 mm right of the midline, and 6.6–7.0 mm deep from the dorsal surface of the cerebellum) according to the atlas of Paxinos and Watson [30]. Following the microinjection, the muscle layers covering the fourth ventricle were sutured. Body temperature was maintained at 37 °C with heating pads until the animals had recovered from surgery. The rats were allowed to recover in their home cages with free access to food and water. Only animals that showed progressive weight gain after the gene transfer were used in subsequent experiments. Before the microinjection of virus into the RVLM, the sites were identified by monitoring BP after local injection of a small dose of L-glutamate.

At the end of the experiment, 2% Pontamine Blue Dye (100 nl) was microinjected to confirm injection site accuracy within the RVLM. The animals were scarified by an intraperitoneal overdose injection of composite anesthetic agents, and the brain was removed. After the rat brain was fixed in 10% formalin for 7 days, frozen brain cross-sections (30 μm) were made and stained with 1% Neutral Red to identify the microinjection sites as in our previous study [9]. The location of each study site was identified and mapped on diagrams of the rat brain (additional file 2: Figure S2 A and B) according to the atlas of Paxinos and Watson [30].

AAV9-mRFP-GFP-LC3/GFP-LC3 was purchased from Hanbio Co. Ltd. Shanghai China. In brief, the novel reporter fusion protein, tandem fluorescent-tagged LC3, mRFP-GFP-LC3 is delivered through an adeno-associated virus (AAV) (Hanbio, Shanghai, China). The GFP signals were attenuated in the lysosomal acidic conditions, whereas mRFP fluorescence was relatively stable after the fusion of autophagosomes with lysosomes. This is because the pKa value of GFP was relatively higher than mRFP (pKa^GFP = 6.0, pKa^mRFP = 4.5). Therefore, this tandem-tagged fluorescent protein showed merged GFP and mRFP fluorescence in the autophagosomes, but only exhibited mRFP fluorescence in lysosomes [31]. All injections were made in a volume of 3 μl within 1 min according to the manufacturer’s instructions. After 2 weeks infection, the animals were scarified and the brain was removed. The section of the RVLM was made to identify the fluorescent dying. Images were taken under a Fluorview FV300 laser scanning confocal microscope (Olympus, Tokyo, Japan). The yellow and red LC3 puncta were manually counted.

At various time intervals after the experimental treatment, rats were sacrificed with an overdose of pentobarbital sodium and perfused intracardially with warm saline. The brain was rapidly removed and immediately frozen on dry ice. the Medulla oblongata covering the RVLM was blocked between 0.5 and 1.5 rostral to the obex, which was adopted from the atlas of Watson and Paxinos [30], and served as the anatomical landmark. These coordinates were selected to cover the extent of the ventrolateral medulla in which functionally identified sympathetic premotor neurons reside. Both sides of the ventrolateral medulla covering the RVLM (approximately at 1.5 to 2.5 mm lateral to the midline and medial to the spinal trigeminal tract) were collected by micropunches with a 1-mm inner diameter burr. There were 1–5 medullary tissues collected from the same experimental groups and the tissues were pooled and stored at −80 °C prior to mRNA or protein analysis.

Total RNA from the RVLM was extracted using an RNA extraction kit RNAqueous (Ambion, TX) according to the manufacturer’s protocol. All RNA isolated was quantified by spectrophotometry and the optical density 260/280 nm ratio was determined. cDNA was synthesized using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, ABI). Following reverse transcription, quantitative real-time PCR was performed on StepOnePlus Real-Time PCR Systems (Applied Biosystems, ABI) under a condition of 40 cycles of 95 °C for 15 s and 60 °C for 1 min, using SYBR Green as indicator. The level of target mRNA was normalized against the expression of the internal control 18S in the same sample that was calculated with the DCt method. The primer pairs for amplification of total IL-1β cDNA (GenBank accession number NM_031512.2) were 5′-GTTACCGTTACTACTCCA T-3′ for the forward primer, and 5′-TGGAGTAGTAACGGTAAC-3′ for the reverse. TNF-α cDNA (GenBank accession number L00981.1) were 5′-GGCGAACCACCAAACG-3′for the forward primer, and 5′-CGTTTGGTGGTTCGCC-3′ for the reverse. β-actin cDNA (GenBank accession number V01217.1) were 5′-ATGGTGGGTATGGGTCAGA-3′ for the forward primer, and 5′-TACCACCCATACCCAGTCT-3′ for the reverse. The expression level of target mRNA in the control group from each independent experiment was considered as 1, and the relative expression level of target mRNA in experimental groups was adjusted as a ratio to its control in each independent experiment and expressed as fold changes.

Total, cytosolic, or mitochondrial protein extract from the RVLM homogenates was used to analyze protein expression by Western blot. In brief, proteins (50 μg for total protein, 20 μg for cytosol, or 10 μg for mitochondria) from the RVLM were separated by using 10–12% SDS-PAGE and transferred to the PVDF membrane. The primary antiserum used for Western blot analysis included a mouse monoclonal antiserum against LC3B (1:1000; Invitrogen, Carlsbad, CA), (1:1000; Wako, Tokyo, Japan), a rabbit polyclonal antiserum against p62 (1:1000; Abcam, Cambridge, UK), TNF-α (1:1000; Calbiochem-Novabiochem), and Iba1 (1:1000; Abcam, Cambridge, UK). This was followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit IgG or goat anti-mouse IgG (Jackson ImmunoReserach, West Grove, PA). Specific antibody-antigen complex was detected using an enhanced chemiluminescence Western blot detection system (NEN Life Science Products, Boston, MA). Cytosolic (LC3-I) and membrane-bound (LC3-II) LC3 proteins were identified based on its molecular size (LC3-I, 18 kDa; LC3-II, 16 kDa). The amount of detected protein was quantified by the Photo-Print Plus software (ETS Vilber-Lourmat, France), and was expressed as the ratio to β-actin protein.

The procedures for double immunofluorescence staining were modified form our previous report [6]. In brief, free-floating 30-μm sections of the medulla oblongata containing the RVLM were incubated with a mouse monoclonal antiserum against LC3B (1:1000; Invitrogen, Carlsbad, CA), a rabbit polyclonal antiserum against p62 (1:1000; Abcam, Cambridge, UK), LAMP2 (1:400; Abcam, Cambridge, UK), NeuN (1:1000; Calbiochem-Novabiochem), and CD11b (1:1000; Cell Signaling, Danvers, MA), which is a specific marker for microglia for 24 h at 4 °C. The sections were subsequently incubated concurrently with a goat anti-rabbit IgG conjugated with Alexa Fluor 488, or a goat anti-mouse IgG conjugated with Alexa Fluor 568. No specific immunoreactivity was observed in control sections, which were incubated without a primary antibody. Viewed under a Fluorview FV300 laser scanning confocal microscope, immunoreactivity exhibited green fluorescence or manifested red fluorescence. The co-localization of red and green fluorescence on merged images indicated the presence of immunoreactivity in neurons or microglia. For quantification of immunoreactive cells in the RVLM, the medullary sections that cover the caudal-to-rostral axis of the RVLM were collected at 105-μm intervals. The immunoreactive cells on both sides of the RVLM from all collected sections were counted by two independent individuals in a single-blind fashion. Total number of counted cells was divided by the number of sections to represent the average number of immunoreactive cells on both sides of the RVLM per section.

Microglia were detected immunohistochemically by the presence of the marker protein CD11b (clone OX-42) which increases markedly upon activation of microglia [32]. Morphological analysis and quantification of microglia was performed with a light microscope using 400 times magnification. In each section, the total number of microglia was counted unilaterally in squares measuring 0.2 × 0.2 mm in size. In areas adjacent to the RVLM, a similar analysis was performed. The numbers of activated microglia counted were expressed as a percentage of the total number of microglia (activated + non-activated) counted in each region analyzed and averaged per section. Counting was performed by a single observer blinded to the treatment. Non-activated microglia were identified by their small soma from which there emanated extensive, highly branched, long, thin processes. Activated microglia were defined by three main criteria [32]: (1) stronger immunohistochemical staining for the marker CD11b, (2) the presence of a clearly enlarged soma, and (3) marked changes in the appearance of the processes which were now reduced in number, but considerably thicker and shorter giving a stubby appearance (Additional file 3: Figure S3).

To investigate the autophagy process, the RVLM were examined via transmission electron microscopy (TEM). The rats in different groups were deeply anesthetized and transcardially perfused with 0.1 M PBS, followed by 4% paraformaldehyde and 1% glutaraldehyde. The RVLM were cut into 1-mm transverse sections and maintained in the same fixative overnight. The sections were subsequently immersed in 1% osmium tetroxide (2 h), dehydrated in graded ethanol, and embedded in epoxy resin. Ultrathin sections (60–70 nm) were obtained using an ultramicrotome; these sections were then post-stained with uranyl acetate and lead citrate and examined using a TEM (Tenai G2 Spirit; Hillsboro, OR, USA).

Tissue levels of glutamate and GABA and plasma levels of norepinephrine (NE) were measured using high-performance liquid chromatography (HPLC) with electrochemical detection as our previous publications [2] and the similar study described [33, 34].

Ionized calcium-binding adaptor molecule-1 (Iba-1) is a calcium-binding protein specifically expressed in macrophage/microglia. Chronic stress increased Iba-1 protein expression, and treatment with minocycline into the cisterna magna attenuated this effect (Fig. 1a). In control rats, there were few microglia (approximately 10%) with an activated morphology in the RVLM. There was significant increase in the proportion of activated microglia in SIH rats compared to the control group. The proportion of activated microglia in the SIH averaged approximately 40–50% (Fig. 1b). Minocycline significantly attenuated the proportion of activated microglia by more than 50%. In the area adjacent to the RVLM, the average number of microglia counted was no significantly different between different groups (Fig. 1b). The morphology of microglia in different groups was shown in Fig. 1c.

Chronic stress significantly upregulated basal levels PICs expression in the RVLM. IL-1β and TNF-α (Fig. 2a) mRNA levels increased 3.8- and 3.0-fold in the RVLM of SIH, respectively. Western blot showed TNF-α protein was increased in SIH rats (Fig. 2b, c). Intracisternal infusion of minocycline could significantly attenuate the upregulation of PICs in the RVLM of SIH rats.

Electric foot shock stressors with noise interventions in rats increased blood pressure and sympathetic vasomotor tone. From the seventh day of the stress exposure, the SBP of the SIH group kept rising gradually until stabilizing around the 14th day (from 109.50 ± 0.77 mmHg to154.83 ± 3.76 mmHg; Fig. S1). In comparison to the control group, animals receiving stressors for 2 weeks induced a significant increase (control vs. SIH, 118.00 ± 2.00 mmHg vs. 154.58 ± 3.32 mmHg, n = 10, P < 0.05) in SBP (Fig. 3a), accompanied by an increase in the sympathetic vasomotor activity reflected by the increase in power density of the LF component (control vs. SIH, 4.1 ± 1.1 mmHg² vs. 5.8 ± 1.2 mmHg², n = 10, P < 0.05) of the SBP spectrum (Fig. 3b). While intracisternal infusion of minocycline for 1 week attenuated stress-induced hypertension and decreased in the sympathetic vasomotor activity (Fig. 3a, b). Microinjection of minocycline or aCSF alone resulted in no apparent change in SBP and the LF component normotensive rats. Changes of body weight, mean atrial pressure (MAP), and HR at the end of the second week of the experiment were shown in Table 1. We observed that SIH rats have higher level of NE (pg/ml) in the plasma than that of control (204.8 ± 13.4 vs. 385.4 ± 29.5, n = 12, P < 0.05), which were attenuated by minocycline microinjection (SIH vs. SIH + Mino 385.4 ± 29.5 vs. 288.4 ± 19.9, n = 10, P < 0.05), suggesting that the sympathetic activity was decreased when the neuroinflammation was inhibited (Fig. 3c). SIH rats showed higher levels of glutamate (Fig. 3d) and lower level of GABA (Fig. 3e) in the RVLM, while minocycline, an inhibitor of microglia activation, attenuated the decrease in GABA and the increases in glutamate of SIH rats (Fig. 3d, e).

Autophagic vacuoles (AVs) existing in the RVLM neurons in vivo was detected by transmission electron microscopy (TEM). Typical AVs contained membrane-bound vacuoles and a mitochondrion [20]. In the control group, few AVs were observed in the RVLM neurons. The normal neurons contain a number of ribosomes, rough endoplasmic reticulum, and mitochondrion (Fig. 4a). An accumulation of AVs (containing mitochondria) and few lysosomal bodies occurred in neurons in the SIH group (Fig. 4b). The number of AVs in control and SIH rats was shown in Fig. 4c.

Western blot showed the autophagosome marker LC3-II/LC3-I ratio was increased (Fig. 5a). Immunofluorescent staining (Fig. 5b) also showed the number of LC3 immuno-positivities was increased (Fig. 5c) in the RVLM neurons of SIH rats. Furthermore, transfected AAV9- -GFP-LC3 dots were coincidently increased in the RVLM neurons of SIH (Fig. 5d).

The concurrent increase of the autophagic marker LC3-II (Fig. 5) and the autophagy selective substrate p62 (Fig. 6) suggested a blockage of autophagic flux. We then monitored the autophagic flux by using the RFP-GFP-tandem fluorescent LC3 (tf-LC3) method. The basis of this method lies in the higher sensitivity of GFP fluorescence to the acidic conditions of the lysosome lumen relative to RFP. The normal RVLM neurons showed some degree of basal autophagic flux as revealed by a few yellow/red dots staining (Fig. 7a). Chronic stress exposure led to an obvious increase in the number of yellow dots per cell (Fig. 7b), indicating the blockage of autophagic flux in SIH. Furthermore, SIH rats showed a decreased expression of lysosomal-associated membrane proteins 2 (LAMP2), which might be one of the causes for the autophagic flux blockage (Fig. 8). Intracisternal infusion of minocycline significantly decreased the yellow dots in the RVLM neurons (Fig. 7a, b) in SIH, which indicated that inhibition of microglia activation in the RVLM facilitated autophagic flux in SIH rats.

Firstly, the relationship between neuroinflammation and neuron autophagic flux was focused currently. Autophagy modulator, e.g., rapamycin bafilomycin A1, chloroquin, or 3-methyladenine (3-MA), on blood pressure was not investigated. Much more experiments in vivo and in vitro will be conducted in future series studies even though we found rapamycin, an autophagy inducer, attenuated the decrease in GABA and the increase in glutamate of SIH rats (Additional file 4: Figure S4). Secondly, we used intracisternal infusion minocycline instead of RVLM microinjection directly because minocycline readily crosses the blood-brain barrier. Increasing doses increase the likelihood of peripheral actions of minocycline, so low dose and chronic administration is requested. It is very hard to chronically implant and fix the minipump in the RVLM. Directly interfering with the neuroinflammtion in the RVLM to observe its effect on autophagy might be essential.