Highlighting the Transition of Mitochondrial Cytochrome C by Immuno-transmission Electron Microscopy

Summary

The protocol describes methods to study micromorphological changes in neurons and the transition of mitochondrial cytochrome c by immuno-transmission electron microscopy.

Abstract

Protein location changes in the microstructure often present a precise regulation mechanism at the level of pathological organelles. How to accurately capture the location of this organelle protein transfer has positive significance and value for the diagnosis and treatment of diseases. Mitochondria play a central role in necrosis and the intrinsic pathway of apoptosis. Under normal conditions, cytochrome c (Cyt c) only exists between the inner and outer membranes of mitochondria and in the nucleus. When cells undergo apoptosis, the mitochondrial inner membrane permeability is altered, and Cyt c is released from mitochondria into the cytoplasm. In this study, a rat model of cerebral ischemia-reperfusion injury was established, and the micromorphological changes of neurons undergoing apoptosis were shown by immuno-transmission electron microscopy. Additionally, it shows the vision of immunogold labeling Cyt c transfer from the mitochondrial matrix to the cytoplasm after cerebral ischemia-reperfusion injury more precisely. The immuno-transmission electron microscopy technique presented in this protocol is suitable for the demonstration of any protein position transitions at the microscopic level.

Introduction

According to the 2021 global burden of disease study, stroke was the 3^rd leading cause of death and the 4^th leading cause of disability-adjusted life-years. There were 11.9 million new stroke events, in which ischemic stroke constituted 65.3% (7.8 million)¹. Neuronal death occurs after cerebral ischemia, including necrosis and apoptosis. The most severely affected brain tissue subsequently undergoes necrotic cell death. Neurons in the ischemic penumbra or periinfarct zone may undergo apoptosis after several hours or days². In addition to bioenergetic and metabolic functions, mitochondria also play a central role in the intrinsic pathway of apoptosis^3,4,5,6.

Cyt C is a protein encoded by nuclear genes. Under normal conditions, Cyt C only exists between the inner and outer membranes of mitochondria and in the nucleus. The apoptotic pathway mediated by its release is associated with the regulatory control of Bcl-2 family members. After being stimulated by apoptosis, Bax/Bak oligomers are formed and inserted into the mitochondrial outer membrane pore, resulting in changes in mitochondrial osmotic pressure and loss of transmembrane potential. Cyt C is released from mitochondria to the cytoplasm, where it binds to the activator of apoptosis 1 to form the apoptotic complex and activate pro-caspase-9. In turn, caspase-3 and caspase-7 are activated, which initiates the Caspase cascade and induces cell apoptosis⁷.

Transmission electron microscopy (TEM) can clearly observe the changes in cell structure at different stages of apoptosis. Electron microscopy morphological observation is a reliable method for determining apoptosis⁸. However, it cannot observe changes in Cyt C in ultrastructure. There are several techniques for detecting specific proteins in ultrastructure: flow cytometry, enzyme-Linked Immunosorbent assay, enzyme-linked immunosorbent assay, spectrophotometry, and Western blotting. However, they cannot accurately capture the location changes of protein in the microstructure. Colloidal gold has particles of different sizes, and the electron density of colloidal gold is high. Immunogold technology is particularly suitable for single or multi-label localization studies of immuno-electron microscopy. Due to its inherent light-to-dark red coloration, colloidal gold is also amenable to observation under light microscopy. When specific proteins are Immunogold labeled, the location changes can be visualized by means of a TEM at a subcellular resolution⁹.

This protocol describes highlighting the transition of mitochondrial Cyt C by immuno-transmission electron microscopy (IEM). The overall goal of this method is to visualize the subcellular translocation of Cyt C during ischemia-induced apoptosis usingIEM. The rationale for IEM is to label Cyt C with colloidal gold and visualize its localization changes at subcellular resolution using TEM⁹. IEM can observe the dynamic changes of proteins at the spatial level, providing a powerful means for explaining physiological and pathological activities¹⁰.

Protocol

The animal program has been approved by the Ethical Committee of SLAS (IACUC Issue No: SYST-2024-018). Here, 6 pathogen-free male SD rats (200-220 g) were raised in independent ventilation cages, maintaining the laboratory environment at 22-25 °C and 45%-65% relative humidity. All rats underwent adaptive feeding for 1 week with free water and diet during the period.

1. Establishment of middle cerebral artery occlusion (MCAO) model

1. Randomly divide the rats into the MCAO group and the sham operation group, with three rats in each group. See Figure 1 for the experimental flowchart.
2. Verify the equipment involved in this protocol according to the list in Figure 2.
3. To establish a model of MCAO of rat ischemia-reperfusion injury, use the intraluminal suture procedure.
4. Fast the rats for 12 h before surgery with water accessible.
5. Anesthetize SD rats with 2% isoflurane (see Table of Materials) in an air-oxygen mixture at 0.6 L/min. Confirm proper anesthetization by clipping the skin of the limbs with forceps; rats should have no retraction or leg reflex. Fasten the thoroughly unconscious rats on the operating table and maintain the body temperature at 37 °C using an animal temperature maintainer. Tape their eyes closed during the surgery to prevent dryness.
6. Remove the fur around the neck with an electric shaver and disinfect the skin with betadine. Make a 1.5 cm midline neck incision with surgical scissors (see Table of Materials), perform blunt dissection on muscle and perivascular fascia, and expose the trachea to locate the right common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA). Surgical tools should be sterilized to prevent infection.
7. Ligate the distal end of the ECA with a 6-0surgicalsuture and place another surgical suture loosely around the proximal end of the ECA to the bifurcation point. Take care not to harm the vagal nerve.
8. Clip the CCA and ICA with vascular clips and make a small incision in the ECA between two surgical sutures.
9. Introduce the monofilament suture into the ECA lumen down toward the CCA, tighten the surgical suture around the ECA stump to secure the intraluminal suture and prevent bleeding. Ensure the monofilament suture matches the weight of the animal to minimize damage and achieve a target infarct volume of 40% in the ipsilateral hemisphere.
10. Cut the remaining portion of the ECA to free the stump and position the ECA stump below the bifurcation point such that it is roughly in line with the ICA.
11. Remove the microvascular clip from the ICA, continue advancing the suture until a mild resistance is felt, indicating the occlusion of the middle cerebral artery; this length is typically around 2 cm. Tie the suture around the monofilament tightly. Remove the other microvascular clip from the CCA.
12. Close the incision with surgical suture and put the rat in a recovery cage, take care of the animal for up to 2 h post-ischemia.

2. Restoration of middle cerebral artery blood flow

1. Before re-anesthesia, check the neuroscore with the Longa Score Scale to evaluate the success of the surgery as described in step 3.
2. Anesthetize the rat as above and reopen the incision. Place a microvascular clip on the CCA, withdraw the occluding suture partway from the ICA until the suture end is visible through the ICA.
3. Place another micro clip on the ICA above the location of the intraluminal suture, completely remove the occluding suture, and tie off the ECA stump. Reperfusion for 24h.
4. Remove the clips from the CCA and ICA. Close the incision with a surgical suture. Administer buprenorphine (0.05 mg/kg; see Table of Materials) subcutaneously.
5. Put the rat back into the cage, provide water and mashed food, and take care until it regains consciousness. Maintain the body temperature of the rats at 37± 0.5 °C during the whole experimental procedure.

3. Assessment of neuroscore

1. Score neurological deficits after the operation and 24 h post-reperfusion using the Longa Score Scale¹¹: (0) no deficit; (1) failure to extend left (paralyzed side) forepaw fully; (2) circling to the left (paralyzed side); (3) falling to the left (paralyzed side); (4) no spontaneous walking and decreased consciousness; (5) dead.
   NOTE: Three operators were evaluated according to the above standards.

4. Brain tissue collection

1. Collect brain tissues 24 h post-reperfusion after neurological deficits are scored and assessed.
2. Anesthetize the rat as described above. Cut the skin to expose the head and neck and cut the cervical cord from the cervical spine with surgical scissors (see Table of Materials).
3. Separate and remove the posterior neck muscles. Carefully remove the skull with curved forceps (see Table of Materials). Pay attention to the dura when separating the skull to avoid scratching the brain tissues. Remove the brain tissues completely.
4. Place the brain tissues in TEM and IEM fixative precooled at 4 °C, and store in the refrigerator at 4 °C.

5. Sham operation

1. For sham operations, perform all procedures as above except that the occlusion is not done.

6. Ultrathin section preparation and imaging

1. TEM ultrathin section preparation
   1. Fixation: Determine the sampling site of fresh tissue to minimize mechanical damage such as traction, contusion, and extrusion, and ensure the tissue thickness does not exceed 2 mm. Ensure the tissue volume does not exceed 1 mm x 1 mm x 1 mm.
   2. Fix with TEM fixative at 4 °C for 24 h. Wash 3x for 15 min each in 0.1 M phosphate buffer PB (pH 7.4; see Table of Materials).
   3. Post fixation: Add 1% osmic acid·0.1 M PB (see Table of Materials) for 2 h at room temperature (25 °C).
   4. Dehydration: Dehydrate the tissue successively in 30% ethanol for 10 min, 50% ethanol for 10 min, 70% ethanol for 15 min, 90% ethanol for 15 min, absolute ethanol for 15 min, and 100% acetone for 15 min.
   5. Gum dipping: Immerse tissues successively in solution epoxy resin SPI-Pon 812: acetone=1:3 for 30 min, solution SPI-Pon 812: acetone = 1:1 for 30 min, and solution SPI-Pon 812: acetone = 3:1 for 1 h (see Table of Materials).
   6. Polymerization and embedding: Immerse the sample in pure SPI-Pon 812 overnight at 37 °C, change out to fresh SPI-Pon 812 for 8 h, and polymerize at 60°C for 48 h (see Table of Materials).
   7. Sectioning: Slice the embedded block semi-thinly at 0.8 µm thickness with a diamond knife, stain with aminotoluene blue at 25 °C for 5 min. Locate and select areas with densely packed neurons with a light microscope for high-magnification observation. Make ultrathin sections with a microtome at 90 nm thickness. Put the samples on 200-Mesh formvar-coated copper grids (see Table of Materials).
   8. Post-staining: Stain grids in uranyl acetate at 25 °C for 15 min, then stain grids in lead citrate at 25 °C for 5 min.
2. IEM ultrathin section preparation
   1. Perform step 6.1.1-6.1.4.
   2. Gum dipping: Immerse tissues successively in solution L. R. White Resin: Ethanol = 1: 3 for 2 h, solution L. R. White Resin: Ethanol =1: 1 for 2 h, and solution L. R. White Resin: Ethanol = 3: 1 for 2 h.
   3. Polymerization and embedding: Immerse the sample in pure L. R. White Resin overnight at 4 °C, change out to fresh L. R. White Resin for 4h at 25 °C and polymerize at 55 °C for 78 h.
   4. Sectioning (as done in step 6.1.7): Slice the embedded blocks semi-thinly at 0.8 µmthickness, stain with aminotoluene blue at 25 °C for 5 min. locate and select areas with densely packed neurons with a light microscope. Make ultrathin sections at 90 nm thickness. Put the samples on copper grids.

7. Immunogold labelling

1. Rinse 2x with PBS and 1x with glycine buffer at 25 °C for 5 min each, then add 2% phosphate buffer of bovine serum albumin (PBS-BSA) and incubate for 30 min.
2. Incubate in the primary antibody (Rabbit polyclonal antibody to Cytochrome C) solution at 37 °C for 2 h. The primary antibody is diluted with PBS-BSA at a ratio of 1:50.
3. Rinse with PBS at 25 °C for 5x for 5 min each. Incubate in the colloidal gold-labeled secondary antibody (Gold-conjugated Goat anti-rabbit IgG) solution at 25 °C for 1 h. The secondary antibody is diluted with PBS-BSA at a ratio of 1:20 (see Table of Materials).
4. Rinse with PBS at 25 °C for 5x for 5 min each. Rinse with distilled water at 25 °C for 5x for 5 min each (see Table of Materials).
5. Post-staining: Stain grids in uranyl acetate at 25 °Cfor 10 min, then stain grids in lead citrate at 25 °C for 2 min (see Table of Materials).
6. Image the ultrathin section and take photos on a transmission electron microscope at 80 kV.

Results

The neuroscore evaluation confirms the success of MCAO, according to Longa Score Scale, rats in MCAO group presented at least circling to the left (neuroscore 2). Rats of the sham operation group showed no obvious neurological dysfunction (neuroscore 0). No mortality was observed during the surgery day.The animals' neuroscore are listed in Table 1.

IEM analysis of the MCAO group revealed neurons with shrunken nuclei and elevated cytoplasmic electron density, accompanied by par...

Discussion

The primary objective of reperfusion therapy in acute ischemic stroke is to restore blood perfusion to the ischemic region. Now we can achieve this goal through intravenous thrombolysis, endovascular treatment, and antithrombotic therapy. However, restoration of blood perfusion leads to ischemia-reperfusion injury, aggravates neuronal necrosis and apoptosis, leading to neurological impairment. During ischemia-reperfusion, abnormal leakage of electrons from the mitochondrial electron transport chain leads to the productio...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.9% normal saline	Sichuan Kelun Pharmaceutical Co., Ltd.	H20083400
200 Mesh formvar coated copper grids	Zhongjingkeyi Technology Co., Ltd.	BZ10262a
220-230g MCAO Monofilaments	Beijing Cinontech Co., Ltd.	A4-243250
Absolute ethanol	Chengdu Kelong Chemical Co.,Ltd.	2023101201
acetone	Chengdu Kelong Chemical Co.,Ltd.	2023120501
Animal anesthesia system	Rayward Life Technology Co., Ltd.	R500
Animal temperature maintainer	Rayward Life Technology Co., Ltd.	69020
Baking table	Shenzhen Bangqi Chuangyuan Technology Co., Ltd.	CT-946
Bone shear	Fine Science Tools	16102-11
capsular embedding molds	Zhongjingkeyi Technology Co., Ltd.	GP130-B
Centrifuge	SCILOGEX	D3024R
Conventional bright-field optical microscopy	Zeiss (Germany)	Primo Star
Curved ophthalmic forceps (10 cm)	Shanghai Medical Equipment Co., Ltd.	211001
Diamond knife	Diatome (Switzerland)	DU4530
Dissecting forceps	Rayward Life Technology Co., Ltd.	F12018-10
Dissecting forceps	Rayward Life Technology Co., Ltd.	F12010-10
Electric blast drying oven	Shanghai YiHENG Scientific Instrument Co., Ltd.	DHG-9030A
Electric shaver	Yiwu Kemei Electric Appliances Co., Ltd.	KM-PG5002
Fixation fluid	Sichuan Scientist Biotechnology CO., Ltd.	SC0001
glass slide	Nantong Mevid Life Science Co., Ltd.	220518001
Gold-conjugated Goat anti-rabbit lgG	Sangon Biotech (Shanghai) Co.,Ltd.	D111094
Hemostatic forceps (12.5 cm)	Anqisheng Biotechnological Co., Ltd.	PL068
Hemostatic forceps (14 cm)	Anqisheng Biotechnological Co., Ltd.	PL002
Isoflurane	Rayward Life Technology Co., Ltd.	2024071201
L. R. White Resin	Tianjin Tainuo Science and Technology Co., Ltd.	AGR1281A
Lead citrate	SPI (US)	GA10701
Needle holder	Jiachi Medical Technology Co., Ltd.	10044036898028
Osmium tetroxide	TED PELLA (US)	4008-220501
PBS	Servicebio	G4202
Rabbit polyclonal antibody to cytochrome c	Affinity Biosciences, Ltd.	AF0146
Rats	Beijing Huafukang Biotechnology Co., Ltd.	110322241102187881
SPI-Pon 812 embedding resin kit	SPI(US)	GS02660
Stereomicroscope	Sunny Optical Technology Co., Ltd.	Soptop SZMN
Straight headed scissors (10 cm)	Anqisheng Biotechnological Co., Ltd.	PL063
Straight ophthalmic forceps (10cm)	Shanghai Medical Equipment Co., Ltd.	211001
Surgical scissors	Rayward Life Technology Co., Ltd.	S14001-10
Transmission electron microscopy	Hitachi (Japan), Ltd.	HT7800
ultramicrotome	Leica(Germany)	UC7
Uranium acetate	SPI (US)	GS02624