What is spinal nerve ligation model?

Spinal cord of rats with neuropathic pain caused by spinal nerve ligation

Relationship between astrocyte activation and pain behavior

three

Zhao? Red? Where's ginger? Sun? Money? Sun Xinyafei? Liu Fengyu? Shi Yushun? Wan? Have △

(Peking University Institute of Neuroscience, Department of Neurobiology, Key Laboratory of Neuroscience, Ministry of Education, Beijing 100083)

Abstract? Objective: To study the relationship between activation of astrocytes in spinal cord and pain behavior in L5 spinal nerve ligation model rats. Method:

44 male SD rats 180 ~ 220? G, randomly divided into 4 groups: sham operation group, spinal nerve ligation 1 day, 3 days and 7 days respectively.

Feng's behavioral use? Frey. The changes of 50% foot contraction threshold of rats at the above time points were measured by hair (? n？ =? 8)? , astrocyte

Immunohistochemical method was used to observe its specific marker GFAP (? n？ =? 3)? . Result:? (1) spinal nerve node

After ligation 1 day, animals developed mechanical pain hypersensitivity. The pain behavior was stable and persistent on the 3rd and 7th day. There was no significant change in sham operation group.

(2) 65 438 days after operation, astrocytes in the spinal dorsal horn of the ligated side were activated. Strong activation of astrocytes was observed on the 3rd and 7th day.

Reaction, the sham operation group can also see a slight activation. (3) Astrocytes were activated after spinal nerve ligation, and the activation degree was similar to that of spinal nerve ligation.

The generation and maintenance of painful behavior are closely related. Conclusion: Activation of astrocytes in spinal dorsal horn may play a role in neuropathic pain.

Use.

Key words? Astrocyte; Glial acidic protein; Neuropathological pain; Spinal nerve ligation

That? Relationship? Between? That? SP？ INAL？ Astrocyte? Activate? And then what? Nerve 2

Sick? Dad? Are you online? Behavior? Follow? ING？ Spine? Nerve? l？ IGATION

Zhao? Hiro? j？ Ang Yu2Qiu，？ Sun? Where is the money? Sun? Yan2Ni，？ l？ IU？ Whispering? Shh? Me? Yu2Shun，？ Wan? you

(Neuroscience? Research? Institute? And then what? Department? Neurobiology? Peking University? Keys? The lab? forNeu2

Ross Sean? Minister? Yes? Education? Beijing? 100083)

Abstract? Goal:? Where to? Investigation? That? Relationship? Between? That? sp？ inal？ Astrocyte? Activate? And then what? Nerve 2

Sick? Pain? Behavior? Are you online? L5？ sp？ inal？ Nerve? Ligation? (? SNL)？ rat ? Method:? 44? A man? SD？ Rats? Used to be. casually

Divided? Become? 4? Group? s:？ Fake? SNL？ 1? d，？ SNL？ 3? d？ And then what? SNL？ 7? d？ Group? s？ Animal mechanics? Abnormal pain? be

Representative? Disgust? By who? 50%? Paws? Abstinence? Threshold? (50%? PWT)？ What is that? Evaluation? With what? Feng? Frey. Filament? (? n

=? 8).？ Use? Immunohistochemistry? Technique? GFAP？ Used to be. Checked? Where to? Evaluation? That? Activate? Yes? astro2

Cells? (? n？ =? 3).？ Result:? ( 1)? That? Mechanical? Abnormal pain? Yes? That? Left? hind？ Paws? Appeared? Open? Day? 1? After that? SNL，

And then what? Continuous? Ever existed? From where? Day? 3? Where to? Day? 7.？ No? Significantly? Change? Mechanical? Abnormal pain? Used to be. observe

Are you online? Fake? Group. ? (2)? Are you online? That? ip？ Side? (? Lesions? Side)? Lumbar spine? Back? Horn? GFAP？ What is immune activity? ob2

Delivered? Where is it? Day? 1.？ This? Kind? Yes? Astrocyte? Activate? Maintenance? Intense? Where is it? Day? 3? And then what? 7,? But? Answer? Slight? degree

Yes? Astrocyte? Activate? Used to be. Observed? Are you online? Fake? Group. ? (3)? Astrocytes? Used to be. Activate? Are you online? That? sp？ inal？ Flexible cord? exist

SNL？ Rats? That? Degree? Yes? Astrocyte? Activate? Related? All right. That? Development? And then what? Maintenance? Yes? neu2

ropathic？ Pain. ? Conclusion:? Astrocyte? Activate? Are you online? That? sp？ inal？ Back? Horn? Is it possible? p？ Lying down? Answer? Role? Are you online? buggy

Pain.

Keys? Words? Astrocyte; ? Glue? Fibrous? Acidity? p？ Protein? (GFAP)？ ; ? Psycho? Pain; ? Sp？ inal？ Nerve? ligate

(? SNL)

3? National Natural Science Foundation of China (30330230, 30570566) and Beijing Natural Science Foundation (7052039)

△ Communication author

China Journal of Pain Medicine? Diary? Yes? Painkillers? 2007,? 13,? ( 1)? 39

& copy; ? 1994-2007? China? Academic? Diary? Electronic? Publishing? house ? All of them? Right? Keep. ? ki.net

Neuropathological pain (? Psycho? Pain) refers to the central or external.

Pain syndrome caused by peripheral nervous system injury or disease is spontaneous.

Sexual pain (? Spontaneous? Pain) Hyperalgesia (? Hyperalgesia)

And hyperalgesia? Abnormal pain) is characterized by. In recent years, glial cells

Activation becomes a new bright spot in the field of pain research [? 1? ]? . In this experiment, L5 was used.

Spinal nerve ligation (? sp？ inal？ Nerve? Ligation? As a mouse of God

The changes of mechanical pain sensitivity in SNL rats were determined by pathological pain model.

And activation of astrocytes in spinal dorsal horn.

To explore the activation of astrocytes in neuropathic pain rats and animals.

For the relationship between.

Materials and methods

1.？ Animal grouping and model preparation

Male SD rats were provided by peking university health science center Experimental Animal Center.

Yes, weight 150 ~ 180? G. They were randomly divided into four groups, namely artificial hands.

Operation group, SNL? 1? Oh, my god. SNL 3? Tianhe SNL? 7? Japanese group. Each group 1 1

Only eight are used for behavioral testing. 3 only used in immunohistochemistry.

Learning analysis.

According to Kim? And the clock [? 2? ] How to make SNL? Model. big

Rats were exposed to chloral hydrate (300? Mg/kg,? Me? p？ ) After anesthesia, put it in the abdomen.

Position, incision of the skin on the back, blunt separation of the left paraspinal muscle, and exposure of L5.

Ligate spinal nerve with No.6 silk thread, stop bleeding, suture and inject.

Penicillin can prevent infection. The operation of sham-operated rats is similar to that of operated rats.

Same, but no nerve ligation.

2.？ Determination of mechanical pain sensitivity in animals

A journalist? Lan, wait [? 3? 】 Report "Up? And then what? Down "method, using

Feng? Frey fiber (? Stoelting Company, USA) was used to measure the hind feet of rats.

50% Foot Contraction Threshold. 15? G is the maximum 50% shrinkage that can be determined by this method.

Foot threshold? 0.？ 25? G is the minimum 50% shrinkage threshold determined by this method.

When the threshold of 50% foot contraction is less than 4? G is considered to be pain sensitivity. Preoperative and

1 day, 3 days and 7 days later, 50% of sham operation group and operation group were measured.

Foot contraction threshold.

3.？ Immunohistochemical experiment

After 1 day, 3 days and 7 days, 10% chloral hydrate (300

Mg/kg,? Me? p？ After anesthesia, open the chest and perfuse the left ventricle. take

The swelling segment of lumbar spine was sliced continuously on a cryostat, and the slice thickness was 14.

Micron.

Hydrate spinal cord slices, inactivate endogenous catalase, and seal.

Get close and join GFAP? Polyclonal antibody (Dako company)? 1? :? 750,

After overnight at 4℃, biotin-labeled secondary antibody (? SP2900 1，

Zhongshan)? Horseradish peroxidase labeled streptavidin (? SP2900 1，

Zhongshan)? , 3,? Color development and gradient dealcoholization of 3'2 biphenyl diamine (DAB).

Water, xylene transparent, sealed and photographed under a microscope.

4.？ statistical treatment

The soles of animals with 50% behavior are calculated according to the above formula.

Foot contraction threshold, immunohistochemical Metamorph software was used to study the ligation side.

The average gray value of the superficial layer of spinal dorsal horn was analyzed, and each spinal cord specimen was selected.

Take five slices and find the average of their gray levels. Statistical analysis of data

With a prism? 4.？ 0 software, the result is the average standard error.

(mean? s？ e？ m)？ Express delivery. Single factor analysis was used to analyze the differences between the data of each group.

Analysis of variance (1? Way? ANOVA) Followed by Dunnett? post2

Ad Hoc test, with p? & lt？ 0.？ 05 as the inspection standard.

result

1.？ After spinal nerve ligation, 50% of the left plantar contraction of rats is subthreshold contraction.

autumn

After spinal nerve ligation 1 day, 50% of the soles of rats contracted at the ligation side.

The threshold is obviously lower than the preoperative basic value, that is, the animal appears mechanical.

Hyperalgesia? After 3 days and 7 days, the pain behavior was stable and persistent (compared with

Compared with normal animals before operation, P? & lt？ 0.？ 0 1)? . Animal feet in sham operation group

The threshold of 50% foot retraction has no significant change compared with the preoperative basic value (compared with surgery)

Compared with normal animals, P? & gt？ 0.？ 05, as shown in figure 1)? .

Figure 1? Changes of mechanical hyperalgesia after spinal nerve ligation in rats. Within 7 days after operation, there were a lot of sham operations.

Changes of 50% contraction threshold of ligation lateral foot in rats and SNL model rats.

3? three

:? p？ & lt？ 0.？ 0 1, compared with the normal control group before operation.

Figure? 1 actile？ Abnormal pain? Are you online? Rats? After that? SNL。 ? That? Change? Yes? 50%? hand

Abstinence? Threshold? (50%? PWT)？ Are you online? Fake? And then what? SNL？ Rats? Inside? 7? A few days.

3? three

:? p？ & lt？ 0.？ 0 1,? Contrast? Where to? That? p？ re2operation？ control

2.？ Immunohistochemical method showed GFAP staining in spinal dorsal horn.

Deepening, glial cells are activated.

Using immunohistochemical staining (DAB), we observed false

Surgery (? Sham) and 1 after spinal nerve ligation? d？ (? SNL？ 1d)？ ,? 3? d

(? SNL？ 3d) and 7? d？ (? SNL？ 7d) astrocytes.

(see figures 2A and 2B)? . As can be seen from the figure, SNL? 1? d

40 ? China Journal of Pain Medicine? Diary? Yes? Painkillers? 2007,? 13,? ( 1)

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GFAP staining in the posterior horn of spinal cord was stronger than that in sham operation group. SNL？ three

D and SNL? 7? d？ Compared with sham operation group, GFAP staining was significantly enhanced.

That is, 1 after nerve injury? Astrocytes are activated. three

D and 7? Astrocytes reacted strongly; Unclosed side

This change did not occur in astrocytes. Sham operation group can also be seen.

Astrocytes in the spinal dorsal horn of the ligated side were slightly activated.

We measured the average gray value of the superficial layer of the spinal dorsal horn on the ligation side.

Analysis, using this value to represent the degree of activation of astrocytes (see figure

2C)？ . After spinal nerve ligation 1 day, astrocytes were excited.

Live,? 3? Tianhe No.7? The degree of activation of astrocytes increased gradually.

In addition

Figure 2? Immunohistochemical staining showed the changes of GFAP staining in spinal dorsal horn at different time points after spinal nerve ligation. (a) after spinal nerve ligation 1? d、3？ D and 7? Three-dimensional GFAP immunohistochemical staining of spinal cord

Color. Scale? A bar? =? 100 micron. (b) is an enlarged view of the dorsal horn of the spinal cord at the ligation side in Figure (a). Scale? A bar? =? 50 microns. (c) As shown in Figure (a), quantify the average gray value of the superficial layer of the dorsal horn of the spinal cord on the ligation side.

Analysis.

3? three

:? p？ & lt？ 0.？ 0 1,? Compared with the sham operation group

Figure? 2? GFAP？ Immune reactivity? Are you online? That? sp？ inal？ Back? Horn? After that? sp？ inal？ Nerve? Ligation. ? (1)? Immunostaining? Yes? GFAP？ Are you online? That? sp？ inal？ Back? Horn? Where is it? How many days? 1,? three

And then what? 7? After that? SNL。 ? Scale? A bar? =? 100μm？ (B)？ Two times? Higher urbanization? Are you online? (1)? . ? Scale? A bar? =? 50μm？ (3)? Quantify? Yes? General? Strength? Are you online? this

Shallow? Level? Yes? That? ip？ Side? sp？ inal？ Back? Horn? As? Display? Are you online? (1)? .

3? three

:? p？ & lt？ 0.？ 0 1,? Contrast? With what? That? Fake? group

discuss

1.？ Activation of astrocytes and neuropathic pain behavior

Relationship between

Astrocytes in the central nervous system perform many functions.

Functions, such as participating in the formation of blood-brain barrier, regulating extracellular ions and nerve transmission.

Mass concentration, maintaining the stability of extracellular pH value, etc. Many stimuli

Can activate astrocytes, such as nerve injury, inflammation, nervous system

Infection and various factors causing pain. After the activation of astrocytes,

A series of changes have taken place, such as hyperplasia, hypertrophy and increased GFAP synthesis.

And increase the release of proinflammatory factors. 1994,? Garrison and others [? 4? ]

Through immunohistochemical technique, it was found that in the sciatic nerve compression model

Astrocytes are activated, if MK2 is used? 80 1 block

Abnormal pain conduction can also block the activation of glial cells, glial

There is a strong correlation between cell activation and amplified pain.

It links glial cells with pain. Later, with the deepening of research.

With the deepening of research, people gradually found that glial cells exist in many pain models.

Be activated [? 5~7? ]? . In this experiment, we ligated the spinal nerve.

The reaction of astrocytes at different time points was observed.

Astrocytes were found after spinal nerve ligation 1 day.

To activate? On the 3rd and 7th days, astrocytes were strongly stimulated.

On-site reaction. The activation of astrocytes mainly occurred on the ligation side.

No astrocytes were found in the dorsal horn, unbound side and anterior horn of spinal cord.

Activate. This is consistent with the report in the literature. After nerve injury,

Astrocytes are activated in the nerve projection area [? 8,? 9? ]? .

Spinal cord is a relay station that transmits pain information from the periphery to the brain and sends it out.

The axonal endings of pain primary afferent neurons terminate in the dorsal horn of spinal cord.

At different levels, it occurs on different types of spinal secondary neurons.

Contact. Among them, there are medium diameter myelinated Aδ fibers and small diameter unmyelinated fibers.

Sheath C fibers terminate in the superficial layer of spinal dorsal horn [? 10? ]? These two fibers act as an intermediary.

Dissemination of harmful information. Therefore, the superficial layer of the spinal dorsal horn is considered to be

The part related to pain transmission. Astrocytes appear here.

China Journal of Pain Medicine? Diary? Yes? Painkillers? 2007,? 13,? ( 1)? 4 1

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Activation indicates that these cells may be involved in the transmission of harmful information.

After spinal nerve ligation 1 day, animals showed obvious mechanical pain.

Min? Animals maintained a high level of pain sensitivity on the 3rd and 7th days. wrong

There was no obvious behavior change in the operation group. Activation of astrocytes

The liver also occurred 65438 0 days after spinal nerve ligation. 3 days and 7 days, activated.

The degree is on the rise. The glial cells in the spinal cord include astrocytes.

Glial cells and microglia. According to the existing literature reports [? 1 1? ]? , ridge

Microglia are not activated or slightly activated after nerve ligation, and microglia

Cytoplasmic inhibitors can not reverse the established pain behavior; What about that star?

Glioblasts have a strong activation reaction after spinal nerve ligation,

And parallel to the occurrence and maintenance of pain behavior, suggesting that astrocytes.

Cells may be involved in the formation of pain behavior. Microglia and stars

Glial cells play different roles in pain, and the current view is that

Start the activation of astrocytes to activate microglia,

However, the activation degree of astrocytes is related to the amplification pain.

Maintain relevance [? 12? ]? . Astrocytes and the Generation and Dimension of Pain Behavior

Is the whole process of persistence related to astrocytes or is it only temporary?

Is the activation of cells a cause or a result? Activation of simple astrocytes.

Whether living animals can induce pain sensitivity needs further verification through experiments.

2.？ Possible significance of astrocytes in neuropathic pain

There is a close relationship between astrocytes and neurons, and they are opposite to each other.

The changes of microenvironment around neurons are very sensitive and can be reversed quickly.

Yes Reactive glial cells can release a large number of neuroactive substances.

Qualitative and proinflammatory factors can increase primary afferent nerve endings.

The release of pain transmitters at the tip and the increase of excitability of pain transmitting neurons

Enlarge pain transmission and form a feedback loop with neurons.

Road [? 13? ]? . This shows that glial cells are no longer in the central nervous system.

A static component, but different from neurons.

Interrupt the dynamic process of information exchange. Glial cells pass through glial network

Lateral branches transmit signals, thus complementing neurons and participating in pain transmission together.

Tour guide. A deeper understanding of the relationship between glial cells and pain will be helpful for research.

Drugs for chronic pain provide new targets.