结核性脑膜炎 z-bo 2020-12-09 351

　　Introduction: Tuberculous meningitis (TBM) accounts for approximately 5%-10% of extrapulmonary tuberculosis cases and 1% of all active tuberculosis cases. It is the most deadly form of tuberculosis. The morbidity and prevalence of TBM is relatively high in children under 4 years old and the elderly, especially those with severe tuberculosis and immunocompromised. A recent study showed that approximately 40% of TBM patients with worsening conditions will die within 6 months of diagnosis. Inflammation and brain damage in the brain are the main features of TBM. Rabbit was established in 1923 as an experimental TBM human disease model. The first rabbit model of TBM included subarachnoid injection of human Mycobacterium tuberculosis (presumably Mtb), avian Mycobacterium tuberculosis, and Mycobacterium bovis. The inoculum prepared in saline did not cause any visible reaction after injection. However, 6-15 days after vaccination, nodules develop in the rabbit's meninges, accompanied by TBM symptoms, such as paralysis, and then die. This early study proved the similarities between human TBM and rabbit TBM models in disease progression and immunopathology. Importantly, the clinical and cerebrospinal fluid (CSF) manifestations of the rabbit TBM model are very similar to human diseases, in which lymphocytes are dominant, protein levels are high, and CSF becomes viscous. In addition, basement membrane thickening and hydrocephalus are common abnormalities in rabbit TBM models and human TBM MRI.

　　 Establishment of a human TBM rabbit model

　　Our research team first used highly toxic (HN878) or highly immunogenic (CDC1551) clinical Mtb isolates to intracerebral infections in rabbits to assess the severity of TBM. In this study, the TBM rabbit model originally published by Dacey and Sande was used. In this model, on the first day, the rabbit skull was fixed with four 0.5×2 inch aluminum screws under anesthesia, and ketamine (0.5 mL/kg) and xylazine (3.5 mL/kg) were injected intramuscularly before bacterial inoculation. Place these screws on the frontal, parietal and occipital cortex of the skull. It is about 1 mm from the top of the skull and about 2 mm from the sagittal suture. Use Dura Base dental acrylic to put a half turnbuckle helmet on top of the screws. The mask allows the New Zealand rabbit to be properly placed in the stereotactic frame. On the second day, the animal was sedated by intramuscular injection of ketamine (0.5 mL/kg) and xylazine (3.5 mL/kg), and the animal’s head was firmly placed in the stereotaxic frame using a 0.25×4 inch bolt. The frame helps to penetrate the brain cistern for bacterial inoculation. Insert a 3.5×25 G spinal needle into the brain cistern, and inject 0.3 mL of bacterial inoculum containing approximately 5×105 CFU. At designated time points after infection, cerebrospinal fluid and blood samples were collected for downstream analysis.

　　 The basic limitation of the previous method is a potential secondary infection, which is caused by the surgical operation required to attach the acrylic helmet to the skull. In addition, the animals were sedated multiple times within a short period of time (about 3 days) to place acrylic helmets and inoculate bacteria. The placement of metal screws and acrylic helmets, as well as various sedative effects, can put pressure on laboratory animals. In addition, the stereotaxic device used in this model does not have an adapter to fix the head of the New Zealand rabbit. The key modification in the new method is the use of an adaptor, which is very suitable for a stereotactic frame and can firmly fix the New Zealand rabbit in place for intracisternal injection. This method also avoids multiple sedation treatments, thereby reducing stress on the animal. The method focuses on improving animal welfare by reducing pain and stress.

　　All animal operations, including Mtb inoculation, are carried out in biosafety level 3 (BSL-3) facilities. All the following steps should be performed in a Class II biological safety cabinet (BSC-2). New Zealand rabbits were placed in a separate negative pressure cage, and the cage was placed in a negative pressure chamber. Before conducting animal infection experiments, a biosafety warning sign must be hung on the door to warn others not to enter during the experiment. Personal protective equipment: disposable Tyvek suit, hair cap, goggles, disposable boots and double gloves. Electric air purifying respirators (PAPR) are used for: (a) infected animals; (b) handling infectious suspensions, and (c) autopsy. The rest of the process can use conventional N95 mask.

　　Prepare for animal infection:

　　 1. In the BSL2/BSL3 animal facility, the specific pathogen-free New Zealand white rabbits weighing 2.4-2.6 kg were bred individually for about one week for domestication.

　　2. On the day of infection, the New Zealand rabbit was moved to the BSL3 facility and sedated by intramuscular injection of ketamine (35 mg/kg) and xylazine (5 mg/kg). After sedation, the animals were moved to the operating room through the isolation filter box.

　　3. Use an electric clipper to shave the fur around the occipital bone of the head of the New Zealand rabbit.

　　4. The animal is placed in a stereotactic frame on a stainless steel table to facilitate intracranial puncture.

　　 Preparation of bacterial inoculum:

　　 5. Prepare the Mycobacterium tuberculosis suspension with 1xPBS sterile normal saline in advance, the concentration is about 5×106cfu/mL.

　　6. Inhale the bacterial suspension into a 1 cc syringe in the biological safety cabinet. Use a special safety device to disconnect the needle, which is located next to the animal frame.

　　7. Remove the central guide wire of the 25 G spinal needle and pull the plunger of the 1 cc syringe containing the inoculum back to leave an air barrier of about 0.2 cc between the tip of the syringe and the inoculum. Carefully remove the needle from the 1cc syringe and connect it to the 25g spinal needle correctly, leaving the protective cover on the needle. The syringe with spinal needle and bacterial inoculum is placed in a covered, leak-proof transfer container, and the top of the container is a paper towel soaked with disinfectant. Seal the container and wipe the outside with DeconPLus wipes, and place it next to the stereotactic frame.

　　 8. The New Zealand rabbit should be fixed in a stereotactic frame to facilitate intracranial puncture. Palpate to determine the correct location of the cistern (a depression area about 5 mm in diameter, located below the occipital protrusion). Then disinfect the injection site with 70% ethanol.

　　9. Use a sterile spinal needle mounted on a 1cc syringe to withdraw a CSF (0.3 mL) sample. The process is as follows: remove the protective sheath of the spinal needle and fix the syringe on the needle base, and then gradually advance the needle horizontally in 5 mm increments at the previously determined position. The needle is passed through the soft tissue until the appropriate resistance is felt to disappear. When pulling out the syringe plunger slowly, you can ensure the needle position in the cistern; there is no blood and CSF to confirm the correct positioning (0.05 mL is enough for visualization in the syringe).

　　 10. Take out the syringe and needle, and put the cerebrospinal fluid sample into a 1.5mL microtube in the biological safety cabinet. The used syringe is discarded in a sharp container. Add 20 microliters of sample to a special cuvette with a lid and fill it with buffer solution. Use a Coulter cell counter to measure the number of WBCs.

　　 Bacterial inoculation:

　　11. Take out the spinal needle mounted on a 1cc syringe containing 0.2 mL of the bacterial inoculation suspension prepared in step 7 from the transfer container, and then carefully push the plunger in to remove the tip air. If droplets form on the needle tip of the syringe, wipe the syringe with a cloth with tuberculosis disinfectant.

　　12. Gradually advance the needle horizontally, fix the syringe on the base, and increase the level in 0.5 cm increments in step 9, and then slowly inject the bacterial inoculum within 20-30 s.

　　 13. Carefully remove the empty syringe with the spinal needle and immediately put it into the sharps box.

　　 14. If necessary, gently press gauze or paper towel at the injection site to stop bleeding. Gauze or paper towels are thrown into biohazard waste bags.

　　 15. Take the New Zealand rabbit out of the stereotaxic device, put it into a separate transfer cage with a filter device, and put it into a breeding cage. At this time, they are still under anesthesia.

　　16. After all animals are inoculated with Mtb, use 10% oxychloride bleach to properly disinfect the stereotaxic frame, the lower traction platform and the cart, keep them in contact for 20 minutes, and then rinse the stereotaxic frame.