# Hypobaric Hypoxia Imbalances Mitochondrial Dynamics in Rat Brain Hippocampus

Authors: Khushbu Jain; Dipti Prasad; Shashi Bala Singh; Ekta Kohli
Journal: Neurology Research International
 (2015)

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## Abstract

Brain is predominantly susceptible to oxidative stress and mitochondrial dysfunction during hypobaric hypoxia, and therefore undergoes neurodegeneration due to energy crisis. Evidences illustrate a high degree of association for mitochondrial fusion/fission imbalance and mitochondrial dysfunction. Mitochondrial fusion/fission is a recently reported dynamic mechanism which frequently occurs among cellular mitochondrial network. Hence, the study investigated the temporal alteration and involvement of abnormal mitochondrial dynamics (fusion/fission) along with disturbed mitochondrial functionality during chronic exposure to hypobaric hypoxia (HH). The Sprague-Dawley rats were exposed to simulated high altitude equivalent to 25000 ft for 3, 7, 14, 21, and 28 days. Mitochondrial morphology, distribution within neurons, enzyme activity of respiratory complexes,Δψm, ADP: ATP, and expression of fission/fusion key proteins were determined. Results demonstrated HH induced alteration in mitochondrial morphology by damaged, small mitochondria observed in neurons with disturbance of mitochondrial functionality and reduced mitochondrial density in neuronal processes manifested by excessive mitochondrial fragmentation (fission) and decreased mitochondrial fusion as compared to unexposed rat brain hippocampus. The study suggested that imbalance in mitochondrial dynamics is one of the noteworthy mechanisms occurring in hippocampal neurons during HH insult.


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## Body

## 1. Introduction

Diminished ambient oxygen pressure at high altitude (HA) causes detrimental effects on central nervous system. Neurodegeneration is one of the major predicaments arising through hypobaric hypoxia (HH) [1] predominantly localized to hippocampus [2]. Oxygen and nitrogen free radical generation, energy dissipation, distorted neurotransmission (glutamate excitotoxicity), elevated corticosterone level, apoptosis, and alteration of genetic and proteomic functions are robust correlate of HH-associated neurodegeneration [3, 4]. Previous studies from our lab pointed out that intense HH exposure causes damage to neurons in rat brain hippocampus. But scientific literature has limited information on the effect of HH on essential organelles such as nucleus, mitochondria, and endoplasmic reticulum, in neurons.Mitochondria are vital organelle which plays an elementary role to provide ATP for regular brain functions [5, 6]. Markedly, whereas detrimental alterations in mitochondrial functions are a substantial primary episode in neurodegeneration, [7] whether and how mitochondrial alterations contribute to neuronal death in HH exposed hippocampal neurons is uncertain.Numerous studies have reported that mitochondria are dynamic organelles and constantly undergo fusion and fission with each other [8], a mechanism major function as mitochondrial quality control. Depending on physiological surroundings, mitochondria fuse to exchange mitochondrial content or divide into small mitochondrion to place inside neural spines [9] via the fusion and fission process, which also manages the shape, length, and number of mitochondria. Besides morphology, function and allocation of mitochondria are also affected by dynamics of fusion and fission process [9]. However cellular ion homeostasis, oxidative stress conditions, and genetic integrity affect mitochondrial fission and fusion to a considerable extent [10]. Therefore, alterations in mitochondrial dynamics are also concerned in various neurodegenerative, psychiatric, and neurometabolic disorders [11, 12].In relation to HH, oxidative stress caused rapid and severe impairment of hippocampal mitochondrial morphology in rat brain hippocampus. More recently, study demonstrated alteration of mitochondrial dynamics during neuronal excitotoxicity in vitro [13] but at present no literature data is available on animal work with mitochondrial dynamics. Current study is aimed at determining whether imbalance in mitochondrial fission and fusion occurs in HH exposed rat brain hippocampus by exploring the expression of its key proteins. We have taken a combination of electron microscopy and immunohistochemistry to demonstrate the above hypothesis. The study also elucidates the fault in mitochondrial transport to neuronal processes. These fusion/fission proteins may serve as key molecular targets to preserve neuronal survival after HH exposed brain damage.


## 2. Material and Methods

### 2.1. Chemicals

All the chemicals and kits were purchased from Sigma Chemicals unless otherwise mentioned.


### 2.2. Animal

Experimentally naive adult male Sprague-Dawley rats (n=60, 10/group; age = 3-4 months, body weight = 220–250 g) were used in the study. Animals were housed in cages (46 cm × 24 cm × 20 cm) with two animals per cage at controlled temperature (25 ± 2°C) and humidity (50% ± 5%) with proper sanitized conditions with a 12 h : 12 h light dark cycle. Food pellets (Lipton India ltd, India) and water were providedad libitum. All procedures involving handling of animal were conducted according to standard guidelines for the care and use of animals in neuroscience and behavioral research (National Research Council, 2003) and guidelines of Institutional Animal Ethical Committee. Healthy rats (mentally and physically) were used for studies and selection was done by anxiety scoring with elevated plus maze (data not shown).


### 2.3. Animal Decompression Chamber

Rats were kept in animal decompression chambers (Seven Star, India) connected to a vacuum pump with a controlled air inflow and outflow. The HH was induced by reducing the barometric pressure to 282 mmHg, resulting in a 59 mmHg oxygen partial pressure (equivalent to high altitude of 25,000 ft (7620 mtr) conditions) in specially designed decompression chamber. Rats were divided into six experimental groups as shown in Table1 and further experiments were carried out. The duration of HH was 0, 3, 7, 14, 21, and 28 days. In groups kept for >1 day, the chambers were opened 20 minutes each day for cage cleaning and food and water replenishment. Fresh air was continuously flushed at a rate of 8 L/min to prevent accumulation of CO2 within the chamber. The rate of ascent and descent was maintained at 300 m/min. Each experimental group of rats had its own littermate Cntrl group, which was kept outside the hypobaric chambers but in the same location. During the simulated HH exposure in the decompression chamber, rats were maintained under the same temperature and humidity as the Cntrl group.Table 1
Effect on food and water consumption with weight in rats during HH exposure.
Animal(N = 6/group)
Weight range (gm)
Food pellets consumed  (gm/cage)
Water  intake  (mL/cage)
Cntrl
220–240
120–180
100–120
3 dHH
200–210
80–100
80–90
7 dHH
160–170
60–80
60–70
14 dHH
170–180
80–100
70–80
21 dHH
180–190
100–110
80–90
28 dHH
180–190
120–130
90–100


### 2.4. Mitochondrial Functionality Evaluation

#### 2.4.1. Isolation of Rat Brain Hippocampus Mitochondria

After HH exposure, the rats were sacrificed by cervical dislocation and the hippocampus was immediately removed, rinsed in saline solution, and used for mitochondrial isolation. Differential centrifugation process was used to isolate mitochondria [14]. The rat hippocampus was homogenized in 5 mL homogenizing buffer (225 mM mannitol, 75 mM sucrose, 5 mM HEPES, 1 mM EGTA, and 1 mg/mL BSA, pH 7.4) per 100 mg tissue.The homogenate was centrifuged at 2000 g for 5 min at 4°C. The pellet was discarded and the supernatant was divided into two parts and centrifuged at 12,000 g for 10 min. The pellet containing the mixture of synaptosomes and mitochondria was mixed in 2 mL of homogenization buffer containing 0.02% digitonin to lyse the synaptosomes followed by centrifugation at 12,000 g for 10 min to pellet down both extrasynaptosomal and intrasynaptosomal mitochondria. The mitochondrial pellet is washed twice in the same buffer without EGTA, BSA, and digitonin.


### 2.5. Yield of Mitochondria

Isolated mitochondria were lysed in ice-cold lysis buffer (0.01 M Tris HCl, pH 7.6, 0.1 M NaCl, 0.1 M DTT, 1 mM EDTA, 0.1% NaN3, PMSF, and protease inhibitor cocktail). The homogenate was centrifuged at 10,000 g for 10 min at 4°C and the supernatant was used for protein expression analysis. 15% SDS-PAGE was run with each sample run in duplicate. 20 μL of sample per 100 mg tissue was resolved by SDS-PAGE and transferred to sandwiched nitrocellulose membranes presoaked in transfer buffer (20% methanol, 0.3% Tris, and 1.44% glycine) by semidry transblot module (BIORAD). The transfer of the protein bands to the membrane was confirmed by Ponceau staining. The membrane was blocked with blocking buffer (3% BSA in PBST) for 1 h and washed with PBST (0.01 M phosphate buffer saline, pH 7.4, 0.1% Tween 20). The membranes were probed with anti-MTCO1, mitochondrial marker (Abcam) overnight at 4°C. Subsequently, the membranes were washed with PBST thrice 10 min each and were incubated with specific secondary anti-IgG antibody for 2 h. The membranes were then developed using DAB reagent. The protein expression was quantified by densitometric analysis.


### 2.6. Mitochondrial Integrity

Isolated mitochondria were added with JC-1 dye (0.5 M) (Δψm sensitive dye) (Molecular Probes, Eugene, OR) in buffer (225 mM mannitol, 75 mM sucrose, 5 mM HEPES, and 1 mM EGTA, pH 7.4). The fluorescence emission of JC-1 at 490 nm (excitation wavelength) was observed by fluorometer concurrently at 530 and 590 nm using plate reader (Spectramax M2, Molecular Probes, USA) with ratio of 590/530 which was used as relative Δψm value.


### 2.7. Enzyme Activity of Mitochondrial Respiratory Complexes

#### 2.7.1. Complex I (NADH Ubiquinone Oxidoreductase)

NADH oxidoreductase activity was deliberated in isolated mitochondria by the method of King and Howard, 1967. The reaction mixture contained 0.2 M glycylglycine buffer pH 8.5, 6 mM NADH in 2 mM glycylglycine buffer and 10.5 mM cytochrome c. Reaction was started with the addition of isolated mitochondrial sample in required quantity. Absorbance change was taken after 30 sec interval at 550 nm for 2 min using Perkin Elmer Lambda 20 spectrophotometer. NADH oxidoreductase activity was expressed asμmole of NADH oxidized per minute per milligram of protein. Absorbance index used cytochrome c (reduced minus oxidised) which is 19.2 mM−1 × cm−1 at 550 nm:(1)NADH+H++cytochrome c⇌NAD++reduced cytochrome cmM NADH oxidised=ΔA550×0.0262.


#### 2.7.2. Complex II (Succinate-Ubiquinone Oxidoreductase) Activity

Succinate dehydrogenase activity was calculated by method of King 1967 [15]. The reaction mixture contains 0.2 M phosphate buffer pH 7.8, 1% BSA, 0.6 M succinic acid, and 0.03 M potassium ferricyanide. Mitochondrial sample was added to initiate the reaction. Absorbance change was observed at 420 nm for 2 min by spectrophotometer. Succinate dehydrogenase activity was expressed as μM succinate oxidized per minute per milligram of total protein (estimated by Bradford assay). The reaction is catalysed by succinate dehydrogenase. Consider the following:(2)Succinate+2FeCN63+electron acceptor⟶fumarate+2FeCN62++2H+mM succinate oxidised=0.485×ΔA420nm.


#### 2.7.3. Complex III (Cytochrome Oxidoreductase) Activity

Cytochrome oxidoreductase activity was measured by method of Roy et al. [16]. The activity of complex III was assayed in mitochondrial sample added with 50 mM KH2PO4 buffer, pH 7.4, containing 1 mM EDTA, 50 μM oxidized cytochrome c, 2 mM KCN, and 10 μM rotenone. After the addition of ubiquinol 2 (150 μM), the rate of reduction of cytochrome c was measured at 550 nm.


#### 2.7.4. Complex IV (Cytochrome c Oxidase) Activity

Cytochrome c oxidase activity was calculated using cytochrome c oxidase assay kit spectrophotometrically. In brief, requisite amount of mitochondrial suspension was added (0.1–0.2 mg protein/mL) in 1x enzyme dilution buffer (20 mM Tris-HCl, pH 7.0 containing 500 mM sucrose). The reaction was started by adding 0.2 mM ferrocytochrome c substrate solution and absorbance was read at 550 nm/min immediately and after 5 sec interval till 2 min. The enzyme activity was expressed inμmole of ferrocytochrome c per minute at pH 7.0 at 25°C. €mM between ferrocytochrome c (reduced) and ferricytochrome c (oxidised) is 21.84. Consider the following:(3)Units/mL=ΔA/min⁡×dil×1.1volume of enzyme×21.84.


### 2.8. ADP: ATP

Hippocampus homogenate was prepared with above protocol used for isolation of mitochondria. The homogenate (10% w/v) was centrifuged at 10,000 g for 15 min. The supernatant was collected and used for further estimations. The temperature was maintained at 4–8°C during the sample preparation. The total protein content per 10 mL of the sample was estimated using Bradford assay and all the readings obtained were converted in terms of 100 mg of proteins.ADP/ATP ratio was measured by ADP/ATP ratio assay bioluminescent kit (Abcam) spectrometrically. In brief, ATP monitoring enzyme was mixed with enzyme reconstitution buffer. 90μL of the above reaction mix with 10 μL of tissue lysate was added to 96-well luminescence plate and luminescence was recorded in luminometer. To measure ADP levels, 1 μL of ADP Converting Enzyme was added and again the luminescence was recorded.


### 2.9. TEM Sample Preparation and Imaging

After HH exposure, the animals were anaesthetized and perfused transcardially first by chilled phosphate buffer saline (0.1 M, pH 7.4), followed by fixation using ice-cold 4% paraformaldehyde (dissolved in 0.1 M PBS, pH 7.4). The brain was dissected out from the skull, and CA1 and CA3 regions of hippocampus were diced into small 1 mm pieces and fixed in the TEM fixative (4% PFA, 2.5% glutaraldehyde in PBS) for 1 day at RT. 2% osmium tetroxide water solution was used to postfix it for 30 min. Later, the sample was dehydrated using a series of acetone solutions with increasing concentration, 30%–100%; each washing step lasted for 30 min. Ultrathin sample sectioning, down to ~90 nm, was performed with a diamond knife after embedding with epoxy resin. TEM was conducted on TECNAI instrument operating at 100 kV by technician who was instructed to find 10 representative neurons (per experimental rat specimen) as identified by a typical nucleus and surrounding perikaryon. Approximately 50 neurons were randomly identified, imaged, and analyzed.


### 2.10. Immunohistochemistry, Tissue Processing, and Cell Counting

After a stipulated period of exposure, the animals were anaesthetized and were immediately perfused transcardially first by chilled PBS (0.1 M, pH 7.4), followed by fixation using ice-cold 4% PFA (dissolved in 0.1 M PBS, pH 7.4). The brain was dissected out from the skull and fixed in the same fixative for 1 day at RT. Afterwards, brains were kept in 10%, 20%, and 30% sucrose in PBS till they sank at the bottom following which 30μm thick coronal sections with an OCT medium (Jung, Leica Biosystems) using a cryostat (Leica CM3050S, Germany) were cut. In brief, each section specified with dorsal hippocampal region was separated in 12-well plate washed in 0.1 M PBS, permeabilized with 0.25% Triton X-100 and blocked with 1.5% goat serum for 3 h at RT. Sections were washed in 0.1 M PBS (pH 7.4) for 30 min and treated with 0.3% H2O2 for 30 min to inhibit endogenous peroxidase activity. Following washing with 0.1 M PBS (pH 7.4), sections (six sections/antibody) were individually incubated with rabbit anti-Mfn1, Mfn2, Opa1, phosho-Drp1, Mff, and Fis1 (1 : 200) antibodies for 48 h at 4°C. The sections were then washed and incubated with HRP enzyme conjugated goat anti-rabbit IgG (1 : 100) for 3 h at RT, and for visualization of the reaction sites, the sections were treated for 2-3 min with DAB reagent and H2O2. Finally, the sections were thoroughly rinsed thrice in distilled water, mounted onto gelatin-coated slides, dehydrated in ethanol, and mounted with DPX mounting medium. The anatomical locations of hippocampus were defined based on standard rat brain atlas (Paxinos and Watson, 1986). Immunostained sections were visualized using Olympus BX51 microscope (Olympus, Model BX 51) and the immunoreactive neurons for Mfn1, Mfn2, Opa1 p-Drp1, Mff, and Fis1 were counted by Image J software in six random fields of 0.1 mm2.


### 2.11. Immunofluorescence and Tissue Processing

After following the above protocol, sections with dorsal hippocampal region were collected and separated in 12-well plate, washed with 0.1 M PBS, permeabilized with 0.25% Triton X-100, and blocked with 1.5% goat serum for 3 h at RT and then washed with 0.1 M PBS (pH 7.4) and incubated with mouse anti-COX IV (1 : 200) for 24 h at 4°C. The sections were then washed and incubated with Alexafluor-488 conjugated goat anti-mouse IgG (1 : 100) for 3 h at RT. Subsequently sections were treated with DAPI (Sigma) for 2-3 min. Finally, the sections were thoroughly rinsed thrice in distilled water, mounted onto gelatin-coated slides, and mounted in antifade mounting medium (Invitrogen). Fluorescence images were acquired on fluorescence microscope (Olympus, Model BX 51) and the mitochondrial distribution in CA1 and CA3 region was identified through intensity variation by Image J software in six random fields of 0.1 mm2.


### 2.12. Statistical Analysis

The estimation of various parameters in the six groups, that is, normoxia, 3 days, 7 days, 14 days, 21 days, and 28 days, exposed to HH was carried out on different days and the mean of each set was taken. All the result values are expressed as mean ± SEM. For each assay, values from hexameric samples were averaged to obtain one value point. The biochemical estimations were analyzed by two-way ANOVA. Multiple comparisont-tests were used for post hoc analysis of significant differences. A p value of <0.05 was considered significant and was indicated on the graphs by asterisk (∗) and p values of <0.01 were indicated by two (∗∗) asterisks. All statistical analyses were done in GraphPad InStat version 3.00 for Windows (GraphPad Software, San Diego, California, USA).


## 2.1. Chemicals

All the chemicals and kits were purchased from Sigma Chemicals unless otherwise mentioned.


## 2.2. Animal

Experimentally naive adult male Sprague-Dawley rats (n=60, 10/group; age = 3-4 months, body weight = 220–250 g) were used in the study. Animals were housed in cages (46 cm × 24 cm × 20 cm) with two animals per cage at controlled temperature (25 ± 2°C) and humidity (50% ± 5%) with proper sanitized conditions with a 12 h : 12 h light dark cycle. Food pellets (Lipton India ltd, India) and water were providedad libitum. All procedures involving handling of animal were conducted according to standard guidelines for the care and use of animals in neuroscience and behavioral research (National Research Council, 2003) and guidelines of Institutional Animal Ethical Committee. Healthy rats (mentally and physically) were used for studies and selection was done by anxiety scoring with elevated plus maze (data not shown).


## 2.3. Animal Decompression Chamber

Rats were kept in animal decompression chambers (Seven Star, India) connected to a vacuum pump with a controlled air inflow and outflow. The HH was induced by reducing the barometric pressure to 282 mmHg, resulting in a 59 mmHg oxygen partial pressure (equivalent to high altitude of 25,000 ft (7620 mtr) conditions) in specially designed decompression chamber. Rats were divided into six experimental groups as shown in Table1 and further experiments were carried out. The duration of HH was 0, 3, 7, 14, 21, and 28 days. In groups kept for >1 day, the chambers were opened 20 minutes each day for cage cleaning and food and water replenishment. Fresh air was continuously flushed at a rate of 8 L/min to prevent accumulation of CO2 within the chamber. The rate of ascent and descent was maintained at 300 m/min. Each experimental group of rats had its own littermate Cntrl group, which was kept outside the hypobaric chambers but in the same location. During the simulated HH exposure in the decompression chamber, rats were maintained under the same temperature and humidity as the Cntrl group.Table 1
Effect on food and water consumption with weight in rats during HH exposure.
Animal(N = 6/group)
Weight range (gm)
Food pellets consumed  (gm/cage)
Water  intake  (mL/cage)
Cntrl
220–240
120–180
100–120
3 dHH
200–210
80–100
80–90
7 dHH
160–170
60–80
60–70
14 dHH
170–180
80–100
70–80
21 dHH
180–190
100–110
80–90
28 dHH
180–190
120–130
90–100


## 2.4. Mitochondrial Functionality Evaluation

### 2.4.1. Isolation of Rat Brain Hippocampus Mitochondria

After HH exposure, the rats were sacrificed by cervical dislocation and the hippocampus was immediately removed, rinsed in saline solution, and used for mitochondrial isolation. Differential centrifugation process was used to isolate mitochondria [14]. The rat hippocampus was homogenized in 5 mL homogenizing buffer (225 mM mannitol, 75 mM sucrose, 5 mM HEPES, 1 mM EGTA, and 1 mg/mL BSA, pH 7.4) per 100 mg tissue.The homogenate was centrifuged at 2000 g for 5 min at 4°C. The pellet was discarded and the supernatant was divided into two parts and centrifuged at 12,000 g for 10 min. The pellet containing the mixture of synaptosomes and mitochondria was mixed in 2 mL of homogenization buffer containing 0.02% digitonin to lyse the synaptosomes followed by centrifugation at 12,000 g for 10 min to pellet down both extrasynaptosomal and intrasynaptosomal mitochondria. The mitochondrial pellet is washed twice in the same buffer without EGTA, BSA, and digitonin.


## 2.4.1. Isolation of Rat Brain Hippocampus Mitochondria

After HH exposure, the rats were sacrificed by cervical dislocation and the hippocampus was immediately removed, rinsed in saline solution, and used for mitochondrial isolation. Differential centrifugation process was used to isolate mitochondria [14]. The rat hippocampus was homogenized in 5 mL homogenizing buffer (225 mM mannitol, 75 mM sucrose, 5 mM HEPES, 1 mM EGTA, and 1 mg/mL BSA, pH 7.4) per 100 mg tissue.The homogenate was centrifuged at 2000 g for 5 min at 4°C. The pellet was discarded and the supernatant was divided into two parts and centrifuged at 12,000 g for 10 min. The pellet containing the mixture of synaptosomes and mitochondria was mixed in 2 mL of homogenization buffer containing 0.02% digitonin to lyse the synaptosomes followed by centrifugation at 12,000 g for 10 min to pellet down both extrasynaptosomal and intrasynaptosomal mitochondria. The mitochondrial pellet is washed twice in the same buffer without EGTA, BSA, and digitonin.


## 2.5. Yield of Mitochondria

Isolated mitochondria were lysed in ice-cold lysis buffer (0.01 M Tris HCl, pH 7.6, 0.1 M NaCl, 0.1 M DTT, 1 mM EDTA, 0.1% NaN3, PMSF, and protease inhibitor cocktail). The homogenate was centrifuged at 10,000 g for 10 min at 4°C and the supernatant was used for protein expression analysis. 15% SDS-PAGE was run with each sample run in duplicate. 20 μL of sample per 100 mg tissue was resolved by SDS-PAGE and transferred to sandwiched nitrocellulose membranes presoaked in transfer buffer (20% methanol, 0.3% Tris, and 1.44% glycine) by semidry transblot module (BIORAD). The transfer of the protein bands to the membrane was confirmed by Ponceau staining. The membrane was blocked with blocking buffer (3% BSA in PBST) for 1 h and washed with PBST (0.01 M phosphate buffer saline, pH 7.4, 0.1% Tween 20). The membranes were probed with anti-MTCO1, mitochondrial marker (Abcam) overnight at 4°C. Subsequently, the membranes were washed with PBST thrice 10 min each and were incubated with specific secondary anti-IgG antibody for 2 h. The membranes were then developed using DAB reagent. The protein expression was quantified by densitometric analysis.


## 2.6. Mitochondrial Integrity

Isolated mitochondria were added with JC-1 dye (0.5 M) (Δψm sensitive dye) (Molecular Probes, Eugene, OR) in buffer (225 mM mannitol, 75 mM sucrose, 5 mM HEPES, and 1 mM EGTA, pH 7.4). The fluorescence emission of JC-1 at 490 nm (excitation wavelength) was observed by fluorometer concurrently at 530 and 590 nm using plate reader (Spectramax M2, Molecular Probes, USA) with ratio of 590/530 which was used as relative Δψm value.


## 2.7. Enzyme Activity of Mitochondrial Respiratory Complexes

### 2.7.1. Complex I (NADH Ubiquinone Oxidoreductase)

NADH oxidoreductase activity was deliberated in isolated mitochondria by the method of King and Howard, 1967. The reaction mixture contained 0.2 M glycylglycine buffer pH 8.5, 6 mM NADH in 2 mM glycylglycine buffer and 10.5 mM cytochrome c. Reaction was started with the addition of isolated mitochondrial sample in required quantity. Absorbance change was taken after 30 sec interval at 550 nm for 2 min using Perkin Elmer Lambda 20 spectrophotometer. NADH oxidoreductase activity was expressed asμmole of NADH oxidized per minute per milligram of protein. Absorbance index used cytochrome c (reduced minus oxidised) which is 19.2 mM−1 × cm−1 at 550 nm:(1)NADH+H++cytochrome c⇌NAD++reduced cytochrome cmM NADH oxidised=ΔA550×0.0262.


### 2.7.2. Complex II (Succinate-Ubiquinone Oxidoreductase) Activity

Succinate dehydrogenase activity was calculated by method of King 1967 [15]. The reaction mixture contains 0.2 M phosphate buffer pH 7.8, 1% BSA, 0.6 M succinic acid, and 0.03 M potassium ferricyanide. Mitochondrial sample was added to initiate the reaction. Absorbance change was observed at 420 nm for 2 min by spectrophotometer. Succinate dehydrogenase activity was expressed as μM succinate oxidized per minute per milligram of total protein (estimated by Bradford assay). The reaction is catalysed by succinate dehydrogenase. Consider the following:(2)Succinate+2FeCN63+electron acceptor⟶fumarate+2FeCN62++2H+mM succinate oxidised=0.485×ΔA420nm.


### 2.7.3. Complex III (Cytochrome Oxidoreductase) Activity

Cytochrome oxidoreductase activity was measured by method of Roy et al. [16]. The activity of complex III was assayed in mitochondrial sample added with 50 mM KH2PO4 buffer, pH 7.4, containing 1 mM EDTA, 50 μM oxidized cytochrome c, 2 mM KCN, and 10 μM rotenone. After the addition of ubiquinol 2 (150 μM), the rate of reduction of cytochrome c was measured at 550 nm.


### 2.7.4. Complex IV (Cytochrome c Oxidase) Activity

Cytochrome c oxidase activity was calculated using cytochrome c oxidase assay kit spectrophotometrically. In brief, requisite amount of mitochondrial suspension was added (0.1–0.2 mg protein/mL) in 1x enzyme dilution buffer (20 mM Tris-HCl, pH 7.0 containing 500 mM sucrose). The reaction was started by adding 0.2 mM ferrocytochrome c substrate solution and absorbance was read at 550 nm/min immediately and after 5 sec interval till 2 min. The enzyme activity was expressed inμmole of ferrocytochrome c per minute at pH 7.0 at 25°C. €mM between ferrocytochrome c (reduced) and ferricytochrome c (oxidised) is 21.84. Consider the following:(3)Units/mL=ΔA/min⁡×dil×1.1volume of enzyme×21.84.


## 2.7.1. Complex I (NADH Ubiquinone Oxidoreductase)

NADH oxidoreductase activity was deliberated in isolated mitochondria by the method of King and Howard, 1967. The reaction mixture contained 0.2 M glycylglycine buffer pH 8.5, 6 mM NADH in 2 mM glycylglycine buffer and 10.5 mM cytochrome c. Reaction was started with the addition of isolated mitochondrial sample in required quantity. Absorbance change was taken after 30 sec interval at 550 nm for 2 min using Perkin Elmer Lambda 20 spectrophotometer. NADH oxidoreductase activity was expressed asμmole of NADH oxidized per minute per milligram of protein. Absorbance index used cytochrome c (reduced minus oxidised) which is 19.2 mM−1 × cm−1 at 550 nm:(1)NADH+H++cytochrome c⇌NAD++reduced cytochrome cmM NADH oxidised=ΔA550×0.0262.


## 2.7.2. Complex II (Succinate-Ubiquinone Oxidoreductase) Activity

Succinate dehydrogenase activity was calculated by method of King 1967 [15]. The reaction mixture contains 0.2 M phosphate buffer pH 7.8, 1% BSA, 0.6 M succinic acid, and 0.03 M potassium ferricyanide. Mitochondrial sample was added to initiate the reaction. Absorbance change was observed at 420 nm for 2 min by spectrophotometer. Succinate dehydrogenase activity was expressed as μM succinate oxidized per minute per milligram of total protein (estimated by Bradford assay). The reaction is catalysed by succinate dehydrogenase. Consider the following:(2)Succinate+2FeCN63+electron acceptor⟶fumarate+2FeCN62++2H+mM succinate oxidised=0.485×ΔA420nm.


## 2.7.3. Complex III (Cytochrome Oxidoreductase) Activity

Cytochrome oxidoreductase activity was measured by method of Roy et al. [16]. The activity of complex III was assayed in mitochondrial sample added with 50 mM KH2PO4 buffer, pH 7.4, containing 1 mM EDTA, 50 μM oxidized cytochrome c, 2 mM KCN, and 10 μM rotenone. After the addition of ubiquinol 2 (150 μM), the rate of reduction of cytochrome c was measured at 550 nm.


## 2.7.4. Complex IV (Cytochrome c Oxidase) Activity

Cytochrome c oxidase activity was calculated using cytochrome c oxidase assay kit spectrophotometrically. In brief, requisite amount of mitochondrial suspension was added (0.1–0.2 mg protein/mL) in 1x enzyme dilution buffer (20 mM Tris-HCl, pH 7.0 containing 500 mM sucrose). The reaction was started by adding 0.2 mM ferrocytochrome c substrate solution and absorbance was read at 550 nm/min immediately and after 5 sec interval till 2 min. The enzyme activity was expressed inμmole of ferrocytochrome c per minute at pH 7.0 at 25°C. €mM between ferrocytochrome c (reduced) and ferricytochrome c (oxidised) is 21.84. Consider the following:(3)Units/mL=ΔA/min⁡×dil×1.1volume of enzyme×21.84.


## 2.8. ADP: ATP

Hippocampus homogenate was prepared with above protocol used for isolation of mitochondria. The homogenate (10% w/v) was centrifuged at 10,000 g for 15 min. The supernatant was collected and used for further estimations. The temperature was maintained at 4–8°C during the sample preparation. The total protein content per 10 mL of the sample was estimated using Bradford assay and all the readings obtained were converted in terms of 100 mg of proteins.ADP/ATP ratio was measured by ADP/ATP ratio assay bioluminescent kit (Abcam) spectrometrically. In brief, ATP monitoring enzyme was mixed with enzyme reconstitution buffer. 90μL of the above reaction mix with 10 μL of tissue lysate was added to 96-well luminescence plate and luminescence was recorded in luminometer. To measure ADP levels, 1 μL of ADP Converting Enzyme was added and again the luminescence was recorded.


## 2.9. TEM Sample Preparation and Imaging

After HH exposure, the animals were anaesthetized and perfused transcardially first by chilled phosphate buffer saline (0.1 M, pH 7.4), followed by fixation using ice-cold 4% paraformaldehyde (dissolved in 0.1 M PBS, pH 7.4). The brain was dissected out from the skull, and CA1 and CA3 regions of hippocampus were diced into small 1 mm pieces and fixed in the TEM fixative (4% PFA, 2.5% glutaraldehyde in PBS) for 1 day at RT. 2% osmium tetroxide water solution was used to postfix it for 30 min. Later, the sample was dehydrated using a series of acetone solutions with increasing concentration, 30%–100%; each washing step lasted for 30 min. Ultrathin sample sectioning, down to ~90 nm, was performed with a diamond knife after embedding with epoxy resin. TEM was conducted on TECNAI instrument operating at 100 kV by technician who was instructed to find 10 representative neurons (per experimental rat specimen) as identified by a typical nucleus and surrounding perikaryon. Approximately 50 neurons were randomly identified, imaged, and analyzed.


## 2.10. Immunohistochemistry, Tissue Processing, and Cell Counting

After a stipulated period of exposure, the animals were anaesthetized and were immediately perfused transcardially first by chilled PBS (0.1 M, pH 7.4), followed by fixation using ice-cold 4% PFA (dissolved in 0.1 M PBS, pH 7.4). The brain was dissected out from the skull and fixed in the same fixative for 1 day at RT. Afterwards, brains were kept in 10%, 20%, and 30% sucrose in PBS till they sank at the bottom following which 30μm thick coronal sections with an OCT medium (Jung, Leica Biosystems) using a cryostat (Leica CM3050S, Germany) were cut. In brief, each section specified with dorsal hippocampal region was separated in 12-well plate washed in 0.1 M PBS, permeabilized with 0.25% Triton X-100 and blocked with 1.5% goat serum for 3 h at RT. Sections were washed in 0.1 M PBS (pH 7.4) for 30 min and treated with 0.3% H2O2 for 30 min to inhibit endogenous peroxidase activity. Following washing with 0.1 M PBS (pH 7.4), sections (six sections/antibody) were individually incubated with rabbit anti-Mfn1, Mfn2, Opa1, phosho-Drp1, Mff, and Fis1 (1 : 200) antibodies for 48 h at 4°C. The sections were then washed and incubated with HRP enzyme conjugated goat anti-rabbit IgG (1 : 100) for 3 h at RT, and for visualization of the reaction sites, the sections were treated for 2-3 min with DAB reagent and H2O2. Finally, the sections were thoroughly rinsed thrice in distilled water, mounted onto gelatin-coated slides, dehydrated in ethanol, and mounted with DPX mounting medium. The anatomical locations of hippocampus were defined based on standard rat brain atlas (Paxinos and Watson, 1986). Immunostained sections were visualized using Olympus BX51 microscope (Olympus, Model BX 51) and the immunoreactive neurons for Mfn1, Mfn2, Opa1 p-Drp1, Mff, and Fis1 were counted by Image J software in six random fields of 0.1 mm2.


## 2.11. Immunofluorescence and Tissue Processing

After following the above protocol, sections with dorsal hippocampal region were collected and separated in 12-well plate, washed with 0.1 M PBS, permeabilized with 0.25% Triton X-100, and blocked with 1.5% goat serum for 3 h at RT and then washed with 0.1 M PBS (pH 7.4) and incubated with mouse anti-COX IV (1 : 200) for 24 h at 4°C. The sections were then washed and incubated with Alexafluor-488 conjugated goat anti-mouse IgG (1 : 100) for 3 h at RT. Subsequently sections were treated with DAPI (Sigma) for 2-3 min. Finally, the sections were thoroughly rinsed thrice in distilled water, mounted onto gelatin-coated slides, and mounted in antifade mounting medium (Invitrogen). Fluorescence images were acquired on fluorescence microscope (Olympus, Model BX 51) and the mitochondrial distribution in CA1 and CA3 region was identified through intensity variation by Image J software in six random fields of 0.1 mm2.


## 2.12. Statistical Analysis

The estimation of various parameters in the six groups, that is, normoxia, 3 days, 7 days, 14 days, 21 days, and 28 days, exposed to HH was carried out on different days and the mean of each set was taken. All the result values are expressed as mean ± SEM. For each assay, values from hexameric samples were averaged to obtain one value point. The biochemical estimations were analyzed by two-way ANOVA. Multiple comparisont-tests were used for post hoc analysis of significant differences. A p value of <0.05 was considered significant and was indicated on the graphs by asterisk (∗) and p values of <0.01 were indicated by two (∗∗) asterisks. All statistical analyses were done in GraphPad InStat version 3.00 for Windows (GraphPad Software, San Diego, California, USA).


## 3. Results

To examine the cause of HH induced neuronal damage, we kept the adult rats in animal decompression chamber at the pressure of 282 mmHg which is 4 times less than the atmospheric pressure; rats were stressed but all rats survived for 28 days. It showed that severe HH stress is tolerable but not mortal to rats. Rats became weak with the hypophagia and hypodipsia [17]. Initially food intake was low, 40–50% in 3 dHH and 7 dHH exposed rats compared to control, and water intake also dropped by 40% (Table 1). Although food and water intake increased in rats to HH after 14th day of exposure but still markedly less as compared to Cntrl group.


### 3.1. Yield of Mitochondria

To study the effect of HH on mitochondria, yield of mitochondria per 100 gm hippocampal tissue was measured. A significant reduction of mitochondrial yield by 30% (p<0.05) and 40% (p<0.01) was found in 3 dHH and 7 dHH exposed rats. But a moderate and gradual increase in the yield of mitochondria was observed after 14 dHH, 21 dHH, and 28 dHH (Figure 1).Figure 1
Photomicrograph of immunoblot and densitometric analysis of MTCO1 (mitochondrial marker) protein depict yield of mitochondria decreases in HH exposure. Result showed mean ± SEM forn=6 in each group. “∗” and “∗∗” denote p<0.05 and p<0.01, respectively, compared to Cntrl.


### 3.2. Mitochondrial Integrity

To investigate the integrity of hippocampal mitochondria, mitochondrial membrane potential was measured by JC-1 dye. JC-1 is a unique cationic dye to signal the loss of mitochondrial membrane potential which emits a peak at 530 nm (green) and another at 590 nm (red) when excited at 488 nm according to membrane potential variation [18]. Fluorescence was measured instantaneously after addition of the dye in isolated mitochondrial suspension. JC-1-stained energized mitochondria exhibit a red orange colour for membrane potential of 140–160 mV and red fluorescence for mitochondria with membrane potential up to 190 mV [19]. The 590 : 530 ratio dropped by 25% at 3 dHH and 48.8% at 7 dHH after HH exposure compared to control. Results point out dissipation of mitochondrial membrane potential after incubation with JC-1 dye in the HH exposed group with significant change at 7 dHH group (p<0.01, Table 2).Table 2
Spectrophotometric investigation of mitochondrial membrane potential (Δψm).
Events
590 nm/530 nm ratio
Cntrl
10 ± 0.81
3 dHH
8.4 ±  0.83∗∗
7 dHH
5.9  ±  0.89∗
14 dHH
6.3 ± 0.79∗
21 dHH
7.7 ± 1.3∗∗
28 dHH
8.2 ± 1.2∗
∗ and ∗∗ denotes p<0.05 and p<0.01 respectively compare to Cntrl.


### 3.3. Enzyme Activity of Respiratory Complexes

Spectrophotometric investigation (Table3) of the activities of the complexes after HH exposure revealed decrease in the activity of complexes I, II, III, and IV as compared to Cntrl. The HH exposure resulted in reduction of the enzymatic activity of complex II to 31% at 3 dHH; similarly 35% reduction was observed at 7 dHH. Enzymatic activity of complex I decreases by 20% at 7 dHH, but there was not significant decrease at other time points. Complexes III and IV also show decrease in activity with the HH exposure by 18% and 22% at 7 dHH. HH exposure at other time points does not show much variation for all four complexes in hippocampal mitochondria. Our experimental data infer that HH inhibits ETC activity.Table 3
Effect of HH on specific activity of respiratory complex enzymes.
Serial number
Mitochondrial complexes of electron transport chain
Cntrl
3 dHH
7 dHH
14 dHH
21 dHH
28 dHH
1
Complex I activity(μmole NADH oxidised/mg protein)
76.26 ± 9.2
70.92 ± 6.6∗
64.83 ± 5.7∗
72.13 ± 4.2∗∗
75.15 ± 6.2∗
75.16 ± 8.1∗∗
2
Complex II activity(succinate-CoQ reductase activity/min/mg protein)
51.28 ± 2.7
45.81 ± 3.1∗
42.05 ± 2.6∗
48.18 ± 1.5∗∗
50.57 ± 2.7∗∗
50.78 ± 3.8∗
3
Complex III activity(cytochrome c-CoQ reductase activity/min/mg protein)
85.72 ± 3.1
77.26 ± 6.6∗
74.91 ± 7.2∗
81.42 ± 2.4∗
83.43 ± 4.1∗∗
84.35 ± 5.6∗∗
4
Complex IV activity(nmol reduced cytochrome c oxidized/min/mg protein)
58.44 ± 3.1
56.82 ± 3.5∗
50.94 ± 3.7∗
53.23 ± 2.2∗∗
55.56 ± 1.5∗
57.24 ± 2.7∗
∗ and ∗∗ denotes p<0.05 and p<0.01 respectively compare to Cntrl.


### 3.4. ADP: ATP Assay

Exposure to HH at 7620 m for 3, 7, 14, 21, and 28 days showed significant ADP: ATP changes in hippocampus. The level of mitochondrial ATP had dropped by 30% at 3 dHH and 50% at 7 dHH after HH exposure (Figure2). Increase of ADP/ATP ratio was observed in all other time points but not very significant.Figure 2
Graphical representation depicting ADP: ATP change HH exposed whole hippocampal lysate compared to Cntrl. Result shows mean ± SEM forn=6 in each group. ∗ denotes p<0.05 compared to Cntrl.


### 3.5. Mitochondrial Morphology by TEM

TEM imaging revealed the change in mitochondrial morphology after HH exposure with all time points. Short, round mitochondria with distorted cristae were observed in HH exposed neurons with significant change at 7 dHH exposure. Moderate changes in mitochondrial structure were also observed in 21 dHH and 28 dHH rat brain mitochondria. Quantitative analysis reveals that mitochondria found in Cntrl rat’s hippocampus are 1-2μm in length as compared to 7 dHH and 14 dHH exposed rats, where ≤0.5–1 μm mitochondria are present (Figure 3). CA3 region also showed same type of mitochondrial morphology with HH exposure compared to Cntrl (data not shown).Figure 3
(a) Representative electron micrograph of hippocampal CA1 region shows temporal alteration in mitochondrial morphology and graphical representation showed changes in (b) mitochondrial length and (c) number.∗ denotes p<0.05 compared to Cntrl. Data are expressed as mean ± SD; n=3. Bar depicted in the figures is equivalent to 50 nm, 1 μm, and 100 nm in length.
(a)
(b)
(c)


### 3.6. Expression of Mitochondrial Fusion/Fission Proteins

There was significant decrease of Mfn1, Mfn2, and Opa1 protein expression in different (CA1 and CA3) regions of hippocampus as compared to Cntrl (Figures4(b) and 4(e)). In case of CA1, both Mfn1 and Mfn2 were decreased in all HH exposed groups with significant decrease in 7 dHH by 27.8% and 32.6% in comparison to Cntrl group. In case of Opa1 protein there was decrease of expression in 7 dHH group of rats compared to Cntrl but after 14 dHH slight recovery of expression was observed in CA1 region in comparison to 7 dHH exposure (Figures 4(b) and 4(e)). In case of CA3 expression of Mfn1, Mfn2, and Opa1 showed significant changes only at 3 dHH, 7 dHH, and 14 dHH. Fis1 protein expression showed remarkable increase at 7 dHH and 14 dHH compared to Cntrl in both CA1 and CA3 regions with slight decrease at 21 and 28 dHH compared to 7 dHH but still upregulated compared to Cntrl. Mff showed very minimal expression with Cntrl rats and in HH exposed rat with no change in HH compared to Cntrl in both CA1 and CA3 regions. Phospho-Drp1 increased with HH exposure with significant increase in 7 dHH group in both CA1 and CA3 regions of hippocampus.Figure 4
Representative photomicrograph of temporal alteration in Mfn1, Mfn2, Opa1, p-Drp1, Mff, and Fis1 protein expression due to HH exposure in (a), CA1 (b) expression of proteins in CA1 region and (c) graphical representation showing cell count expressing protein in CA1 region (d) CA3 (e) expression of protein in CA3 regions of rat hippocampus (f) bar graph presents cell count expressing protein in CA3 region. Data are expressed as mean ± SEM;n=3. Scale bar depicted in the figures is equivalent to 10 μm length.
(a)
(b)
(c)
(d)
(e)
(f)


### 3.7. Mitochondrial Distribution

Immunofluorescence of COX IV showed the presence of mitochondria in cell soma of CA1 region of hippocampus. The mitochondrial localization was ascertained from the fluorescence intensity. There is a change in mitochondrial localization with all time points. Mitochondria were observed like dotted structure in whole neuron with cell soma, axons, and dendrites in Cntrl group but were observed restricted to cell soma in 7 dHH, 14 dHH, and 21 dHH (Figures5(a) and 4(b)). It was also observed that the variation in mitochondrial distribution was increased significantly (p<0.01) in 7 days group and 14 days group in comparison to Cntrl. CA3 region presents a similar pattern of distribution of mitochondria in HH exposed rats compared to Cntrl (data not shown).Figure 5
(a) Representative fluorescence micrograph of the temporal alterations of COX IV (mitochondrial respiratory complex IV) distribution and expression in HH exposed CA1 hippocampal region. (b) CA1 region COX IV merged with DAPI. (c) Enlarged view depicts mitochondria in neuron. Figures (i) and (ii) depict that mitochondria are equally distributed in cell soma, axons, and dendrites in Cntrl rat hippocampal tissue and figures (iii) and (iv) depict that reduction in mitochondria and distribution constraints to cell soma only in 7 dHH exposed hippocampal tissue. Yellow colour arrows indicate mitochondria as green dots of high fluorescence intensity. Data are expressed as mean ± SD;n=3. Scale bar 10 μm, 20 μm.
(a)
(b)
(c)


## 3.1. Yield of Mitochondria

To study the effect of HH on mitochondria, yield of mitochondria per 100 gm hippocampal tissue was measured. A significant reduction of mitochondrial yield by 30% (p<0.05) and 40% (p<0.01) was found in 3 dHH and 7 dHH exposed rats. But a moderate and gradual increase in the yield of mitochondria was observed after 14 dHH, 21 dHH, and 28 dHH (Figure 1).Figure 1
Photomicrograph of immunoblot and densitometric analysis of MTCO1 (mitochondrial marker) protein depict yield of mitochondria decreases in HH exposure. Result showed mean ± SEM forn=6 in each group. “∗” and “∗∗” denote p<0.05 and p<0.01, respectively, compared to Cntrl.


## 3.2. Mitochondrial Integrity

To investigate the integrity of hippocampal mitochondria, mitochondrial membrane potential was measured by JC-1 dye. JC-1 is a unique cationic dye to signal the loss of mitochondrial membrane potential which emits a peak at 530 nm (green) and another at 590 nm (red) when excited at 488 nm according to membrane potential variation [18]. Fluorescence was measured instantaneously after addition of the dye in isolated mitochondrial suspension. JC-1-stained energized mitochondria exhibit a red orange colour for membrane potential of 140–160 mV and red fluorescence for mitochondria with membrane potential up to 190 mV [19]. The 590 : 530 ratio dropped by 25% at 3 dHH and 48.8% at 7 dHH after HH exposure compared to control. Results point out dissipation of mitochondrial membrane potential after incubation with JC-1 dye in the HH exposed group with significant change at 7 dHH group (p<0.01, Table 2).Table 2
Spectrophotometric investigation of mitochondrial membrane potential (Δψm).
Events
590 nm/530 nm ratio
Cntrl
10 ± 0.81
3 dHH
8.4 ±  0.83∗∗
7 dHH
5.9  ±  0.89∗
14 dHH
6.3 ± 0.79∗
21 dHH
7.7 ± 1.3∗∗
28 dHH
8.2 ± 1.2∗
∗ and ∗∗ denotes p<0.05 and p<0.01 respectively compare to Cntrl.


## 3.3. Enzyme Activity of Respiratory Complexes

Spectrophotometric investigation (Table3) of the activities of the complexes after HH exposure revealed decrease in the activity of complexes I, II, III, and IV as compared to Cntrl. The HH exposure resulted in reduction of the enzymatic activity of complex II to 31% at 3 dHH; similarly 35% reduction was observed at 7 dHH. Enzymatic activity of complex I decreases by 20% at 7 dHH, but there was not significant decrease at other time points. Complexes III and IV also show decrease in activity with the HH exposure by 18% and 22% at 7 dHH. HH exposure at other time points does not show much variation for all four complexes in hippocampal mitochondria. Our experimental data infer that HH inhibits ETC activity.Table 3
Effect of HH on specific activity of respiratory complex enzymes.
Serial number
Mitochondrial complexes of electron transport chain
Cntrl
3 dHH
7 dHH
14 dHH
21 dHH
28 dHH
1
Complex I activity(μmole NADH oxidised/mg protein)
76.26 ± 9.2
70.92 ± 6.6∗
64.83 ± 5.7∗
72.13 ± 4.2∗∗
75.15 ± 6.2∗
75.16 ± 8.1∗∗
2
Complex II activity(succinate-CoQ reductase activity/min/mg protein)
51.28 ± 2.7
45.81 ± 3.1∗
42.05 ± 2.6∗
48.18 ± 1.5∗∗
50.57 ± 2.7∗∗
50.78 ± 3.8∗
3
Complex III activity(cytochrome c-CoQ reductase activity/min/mg protein)
85.72 ± 3.1
77.26 ± 6.6∗
74.91 ± 7.2∗
81.42 ± 2.4∗
83.43 ± 4.1∗∗
84.35 ± 5.6∗∗
4
Complex IV activity(nmol reduced cytochrome c oxidized/min/mg protein)
58.44 ± 3.1
56.82 ± 3.5∗
50.94 ± 3.7∗
53.23 ± 2.2∗∗
55.56 ± 1.5∗
57.24 ± 2.7∗
∗ and ∗∗ denotes p<0.05 and p<0.01 respectively compare to Cntrl.


## 3.4. ADP: ATP Assay

Exposure to HH at 7620 m for 3, 7, 14, 21, and 28 days showed significant ADP: ATP changes in hippocampus. The level of mitochondrial ATP had dropped by 30% at 3 dHH and 50% at 7 dHH after HH exposure (Figure2). Increase of ADP/ATP ratio was observed in all other time points but not very significant.Figure 2
Graphical representation depicting ADP: ATP change HH exposed whole hippocampal lysate compared to Cntrl. Result shows mean ± SEM forn=6 in each group. ∗ denotes p<0.05 compared to Cntrl.


## 3.5. Mitochondrial Morphology by TEM

TEM imaging revealed the change in mitochondrial morphology after HH exposure with all time points. Short, round mitochondria with distorted cristae were observed in HH exposed neurons with significant change at 7 dHH exposure. Moderate changes in mitochondrial structure were also observed in 21 dHH and 28 dHH rat brain mitochondria. Quantitative analysis reveals that mitochondria found in Cntrl rat’s hippocampus are 1-2μm in length as compared to 7 dHH and 14 dHH exposed rats, where ≤0.5–1 μm mitochondria are present (Figure 3). CA3 region also showed same type of mitochondrial morphology with HH exposure compared to Cntrl (data not shown).Figure 3
(a) Representative electron micrograph of hippocampal CA1 region shows temporal alteration in mitochondrial morphology and graphical representation showed changes in (b) mitochondrial length and (c) number.∗ denotes p<0.05 compared to Cntrl. Data are expressed as mean ± SD; n=3. Bar depicted in the figures is equivalent to 50 nm, 1 μm, and 100 nm in length.
(a)
(b)
(c)


## 3.6. Expression of Mitochondrial Fusion/Fission Proteins

There was significant decrease of Mfn1, Mfn2, and Opa1 protein expression in different (CA1 and CA3) regions of hippocampus as compared to Cntrl (Figures4(b) and 4(e)). In case of CA1, both Mfn1 and Mfn2 were decreased in all HH exposed groups with significant decrease in 7 dHH by 27.8% and 32.6% in comparison to Cntrl group. In case of Opa1 protein there was decrease of expression in 7 dHH group of rats compared to Cntrl but after 14 dHH slight recovery of expression was observed in CA1 region in comparison to 7 dHH exposure (Figures 4(b) and 4(e)). In case of CA3 expression of Mfn1, Mfn2, and Opa1 showed significant changes only at 3 dHH, 7 dHH, and 14 dHH. Fis1 protein expression showed remarkable increase at 7 dHH and 14 dHH compared to Cntrl in both CA1 and CA3 regions with slight decrease at 21 and 28 dHH compared to 7 dHH but still upregulated compared to Cntrl. Mff showed very minimal expression with Cntrl rats and in HH exposed rat with no change in HH compared to Cntrl in both CA1 and CA3 regions. Phospho-Drp1 increased with HH exposure with significant increase in 7 dHH group in both CA1 and CA3 regions of hippocampus.Figure 4
Representative photomicrograph of temporal alteration in Mfn1, Mfn2, Opa1, p-Drp1, Mff, and Fis1 protein expression due to HH exposure in (a), CA1 (b) expression of proteins in CA1 region and (c) graphical representation showing cell count expressing protein in CA1 region (d) CA3 (e) expression of protein in CA3 regions of rat hippocampus (f) bar graph presents cell count expressing protein in CA3 region. Data are expressed as mean ± SEM;n=3. Scale bar depicted in the figures is equivalent to 10 μm length.
(a)
(b)
(c)
(d)
(e)
(f)


## 3.7. Mitochondrial Distribution

Immunofluorescence of COX IV showed the presence of mitochondria in cell soma of CA1 region of hippocampus. The mitochondrial localization was ascertained from the fluorescence intensity. There is a change in mitochondrial localization with all time points. Mitochondria were observed like dotted structure in whole neuron with cell soma, axons, and dendrites in Cntrl group but were observed restricted to cell soma in 7 dHH, 14 dHH, and 21 dHH (Figures5(a) and 4(b)). It was also observed that the variation in mitochondrial distribution was increased significantly (p<0.01) in 7 days group and 14 days group in comparison to Cntrl. CA3 region presents a similar pattern of distribution of mitochondria in HH exposed rats compared to Cntrl (data not shown).Figure 5
(a) Representative fluorescence micrograph of the temporal alterations of COX IV (mitochondrial respiratory complex IV) distribution and expression in HH exposed CA1 hippocampal region. (b) CA1 region COX IV merged with DAPI. (c) Enlarged view depicts mitochondria in neuron. Figures (i) and (ii) depict that mitochondria are equally distributed in cell soma, axons, and dendrites in Cntrl rat hippocampal tissue and figures (iii) and (iv) depict that reduction in mitochondria and distribution constraints to cell soma only in 7 dHH exposed hippocampal tissue. Yellow colour arrows indicate mitochondria as green dots of high fluorescence intensity. Data are expressed as mean ± SD;n=3. Scale bar 10 μm, 20 μm.
(a)
(b)
(c)


## 4. Discussion

In our previous studies, we investigated the effect of long duration of continuous simulated HH, that is, 7, 14, 21, and 28 days, on rat brain hippocampus with respect to modification in neurotransmission and corticosterone levels along with other genetic and proteomic factors. In this study an effort is made to advance the existing literature since association of organelle level disturbance with HH induced neuronal stress is not much studied. Numerous studies support the hypothesis that disruption of mitochondrial function plays a central role in the pathophysiology of many neurological diseases. However, there appears to be no published data on mitochondrial dysfunction with imbalance in its dynamics under long duration HH in rat brain hippocampus to the best of our knowledge.Results from our experiments showed continuous exposure to HH caused alterations in mitochondrial functionality with ETC enzymatic activity leading to reduced ATP generation as well as amended mitochondrial dynamics in time-dependent manner. From the 3rd and 7th days of exposure, the mitochondrial alteration levels were significantly higher than on the 14th, 21st, and 28th days. Our results have clearly pointed out that HH causes imbalance of fusion and fission system along with augmented mitochondrial dysfunction. These results are in agreement with earlier studies which showed that application of HH, hypoxia reoxygenation, and anoxia-reoxygenation imbalances mitochondrial dynamics in various cellular and animal models with targeted organs other than brain [20–22].Besides numerous preceding researches, our results indicated that HH occurs with multiple factors contribution, including hypophagia and hypodipsia as the preliminary observance in HH exposed rats (Table1), which might be due to changes in ghrelin, leptin, adiponectin, and such hormones [17]. Findings also suggest mitochondrial yield was decreased in hippocampus of animals exposed to HH as compared to Cntrl (Figure 1). Decreased yield might be synergistic effect of imbalanced mitochondrial biogenesis [23] and mitophagy due to HH stress. Results describe that HH stress also modulated the Δψm, suggestive of both structural and functional deformity (Table 2). This in turn influenced elements of the energy synthesis processes as complexes I, II, III, IV (Table 3) and ATP/ADP carrier (Figure 2), with the decrease in their activities. Our results are consistent with the study reported earlier [24] that deviated Δψm along with ROS generation may possibly slow down the ETC activity. Decreased ETC activity continues to the reduction in ATP production (Figure 2).Extreme loss and damage of mitochondria as observed in rats exposed to HH in terms of disruption of mitochondria, depolarization of mitochondrial membrane potential, and interruption of mitochondrial functionality should provoke quality control mechanism of mitochondria to maintain the cell survival. To explore that, we investigated the expression of key proteins involved in fusion/fission process mainly mitochondrial fusion (i.e., Opa1, Mfn1, and Mfn2) and fission proteins (i.e., Fis1, Drp1, and Mff) in hippocampal tissues from HH exposed rats to Cntrl rats. Immunohistochemical analysis revealed that all these six proteins documented considerable alteration in HH exposed rat brain hippocampus: Opa1, Mfn1, and Mfn2 levels were reduced by 42.4%, 27.8%, and 32.6%, respectively, in CA1 region (Figure4(b)). Phospho-Drp1 is the active form of Drp1 and shows increase in expression by 21% (Figure 4(b)). Interestingly, unlike other fission/fusion proteins, Fis1 levels were increased significantly, 5.2-fold, on 7 dHH exposure (Figure 4(b)). Mff does not show any expression (Figure 4(b)) in Cntrl and exposed rats might be as Mff which is a functionally similar protein to Fis1 and worked for an alternate fission pathway, therefore not expressed here in rat hippocampus. CA3 region of hippocampus also showed almost similar pattern of expression of fusion/fission proteins (Figure 4(e)). In order to understand mitochondria in detail we have performed EM, of hippocampal tissues targeting the CA1 region, which describes damaged mitochondria with distorted cristae in HH exposed rats in all time points, and the significant increase of short and round mitochondria was observed in 7 dHH and 14 dHH rats which gradually decreases in 21 dHH and 28 dHH. These EM results validate our immunohistochemistry results (Figure 3).We infer, probably, fission proteins endeavour to repair damaged mitochondria, which on imbalance cause excessive fragmentation of mitochondria. Legros et al. [25] suggested that fusion was abolished by dissipation of the inner membrane potential. Consequently we presume that, due to MMP depolarization, fusion is impeded during HH augment rapid (4 h) fragmentation of mitochondria by fission. At later time points, 14 dHH and 21 dHH exposed rats, system tries to balance fusion and fission again. Fragmented and unhealthy (with the dissipated membrane potential) mitochondria undergo mitophagy. That revives the fusion mechanism in 21 dHH and 28 dHH exposed rats. But this revival is not 100% at 28 dHH as in Cntrl.The alteration in mitochondrial allocation in HH exposed hippocampus is also showed by COX IV staining. Although it is weak evidence, immunohistochemical analysis revealed noticeable changes in the distribution of COX IV, which in control cases is seen throughout the soma and processes of neurons of hippocampus, while, in HH exposed rats, the localization is basically restricted to the soma (Figures5(a) and 5(b)). To quantify the changes in COX IV distribution, the immunoreactivity was measured from the top of the neuron, sometimes extending along the axon, and expressed as COX IV labeling distance. COX IV positive mitochondria extend further down the axon in control rats, while, in HH exposed hippocampus, COX IV is often restricted to the perinuclear region (Figure 5(c) (i), (ii), (iii), and (iv)). Our data suggest that changes in the expression of these mitochondrial fission and fusion proteins likely underlie abnormal mitochondrial distribution in HH exposed hippocampal neurons. This indicative study can be extended in future research.Taken together, our data demonstrated immense changes in the expression of mitochondrial fission and fusion proteins and altered mitochondrial dynamics which likely contributes to mitochondrial and neuronal dysfunction in HH induced stress. However, further studies are needed to determine the actual mechanism as to how HH suppresses fusion and what other factors are involved in the mechanism.


## 5. Conclusion

In conclusion we have demonstrated the imbalance of mitochondrial fusion fission proteins in hippocampus leading to neurodegeneration. Fission predominates over fusion during oxygen deprivation and mitochondrial fragmentation occurs in hippocampal neurons. This study suggests mitochondrial dynamics proteins as potential key players to promote neurodegeneration in HH insult.


## Highlights

(i)
Mitochondrial yield and functionality are affected by HH stress.
(ii)
Dynamic process of mitochondrial fusion/fission is also altered by HH in rat brain hippocampus.
(iii)
Fragmented mitochondria with distorted cristae in hippocampal neurons are frequently observed in HH exposed rats.



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*Source: 742059-2015-07-05.xml*