Intravital Imaging of Fluorescent Protein Expression in Mice with a Closed-Skull Traumatic Brain Injury and Cranial Window Using a Two-Photon Microscope

Summary

This study demonstrates delivery of a repetitive traumatic brain injury to mice and simultaneous implantation of a cranial window for subsequent intravital imaging of a neuron-expressed EGFP using two-photon microscopy.

Abstract

The goal of this protocol is to demonstrate how to longitudinally visualize the expression and localization of a protein of interest within specific cell types of an animal's brain, upon exposure to exogenous stimuli. Here, the administration of a closed-skull traumatic brain injury (TBI) and simultaneous implantation of a cranial window for subsequent longitudinal intravital imaging in mice is shown. Mice are intracranially injected with an adeno-associated virus (AAV) expressing enhanced green fluorescent protein (EGFP) under a neuronal specific promoter. After 2 to 4 weeks, the mice are subjected to a repetitive TBI using a weight drop device over the AAV injection location. Within the same surgical session, the mice are implanted with a metal headpost and then a glass cranial window over the TBI impacting site. The expression and cellular localization of EGFP is examined using a two-photon microscope in the same brain region exposed to trauma over the course of months.

Introduction

Traumatic brain injury (TBI), which can result from sports injuries, vehicle collisions, and military combat, is a worldwide health concern. TBI can lead to physiological, cognitive, and behavioral deficits, and lifelong disability or mortality^1,2. TBI severity can be classified as mild, moderate, and severe, the vast majority being mild TBI (75%-90%)³. It is increasingly recognized that TBI, particularly repetitive occurrences of TBI, can promote neuronal degeneration and serve as risk factors for several neurodegenerative diseases, including Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and chronic traumatic encephalopathy (CTE)^4,5,6. However, the molecular mechanisms underlying TBI-induced neurodegeneration remain unclear, and thus represent an active area of study. To gain insight into how neurons respond to and recover from TBI, a method for monitoring fluorescently tagged proteins of interest, specifically within neurons, by longitudinal intravital imaging in mice after TBI is described herein.

To this end, this study shows how to combine a surgical procedure for the administration of closed-skull TBI that is similar to what that has been reported previously^7,8, together with a surgical procedure for implantation of a cranial window for downstream intravital imaging, as described by Goldey et al⁹. Notably, it is not feasible to implant a cranial window first and subsequently perform a TBI in the same region, as the impact of the weight drop that induces the TBI is likely to damage the window and cause irreparable harm to the mouse. Therefore, this protocol was designed to administer the TBI and then implant the cranial window directly over the impact site, all within the same surgical session. An advantage of combining both the TBI and cranial window implantation in a single surgical session is a reduction in the number of times a mouse is subjected to surgery. Further, it allows one to monitor the immediate response (i.e., on the timescale of hours) to TBI, as opposed to implanting the window at a later surgical session (i.e., initial imaging starting on a timescale of days post-TBI). The cranial window and intravital imaging platform also offer advantages over monitoring neuronal proteins by conventional methods such as immunostaining of fixed tissues. For example, fewer mice are required for intravital imaging, as the same mouse can be studied at multiple time points, as opposed to separate cohorts of mice needed for discrete time points. Further, the same neurons can be monitored over time, allowing one to track specific biological or pathological events within the same cell.

As a proof of concept, the neuron-specific expression of enhanced green fluorescent protein (EGFP) under the synapsin promoter is demonstrated here¹⁰. This approach can be extended to 1) different brain cell-types by utilizing other cell-type specific promoters, such as myelin basic protein (MBP) promoter for oligodendrocytes and glial fibrillary acidic protein (GFAP) promoter for astrocytes¹¹ , 2) different target proteins of interest by fusing their genes with the EGFP gene, and 3) co-expressing multiple proteins fused to different fluorophores. Here, EGFP is packaged and expressed via adeno-associated virus (AAV) delivery through an intracranial injection. A closed-skull TBI is administered using a weight-drop device, followed by implantation of a cranial window. Visualization of neuronal EGFP is achieved through the cranial window, using two-photon microscopy to detect EGFP fluorescence in vivo. With the two-photon laser, it is possible to penetrate deeper into the cortical tissue with minimal photodamage, allowing for repeated longitudinal imaging of the same cortical regions within an individual mouse for days and up to months^12,13,14,15. In sum, this approach of combining a TBI surgery with intravital imaging aims to advance the understanding of the molecular events that contribute to TBI-induced disease pathology^16,17.

Protocol

All the animal related protocols were conducted in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Research Council (US) Committee. The protocols were approved by the Institutional Animal Care and Use Committee of University of Massachusetts Chan Medical School (UMMS) (Permit Number 202100057). In brief, as shown in the schematic of study (Figure 1), the animal receives a virus injection, a TBI, a window implantation, and then intravital imaging in a time sequence.

NOTE: Commercial terms have been removed. Please refer to the Table of Materials for the specific equipment used.

1. Intracranial injection of AAV using a stereotaxic device

1. AAV(PHP.eB)-Syn1-EGFP
   1. Use a viral titer of 1 x 10¹³ viral genomes per milliliter (vg/mL). Syn1 refers to Synapsin1, which is a neuronal specific promotor allowing for restricted viral expression in neurons. The virus can be prepared in-house or outsourced.
2. Preparation for stereotaxic injection surgery for administration of AAV
   1. Autoclave regular scissors, a surgical caliper, surgical spring scissors, surgical forceps, mosquito forceps, gauze, a cotton tipped applicator, and a glass microliter syringe.
   2. Sanitize the surgery area using 75% ethanol. Expand the disposable sterile surgical drape to cover the surgery region on the stereotaxic platform.
      NOTE: To maintain animal body temperature during the injection surgery, use a feedback regulated heating apparatus.
   3. Weigh and record the mouse body weight.
   4. Open the oxygen tank valve and adjust the oxygen flow to 1.5 L/min. Open the anesthesia machine and set the isoflurane level value to three.
      NOTE: The carrier gas can be either room air or 100% oxygen in this step.
   5. Put the mouse into the induction chamber and let it stay for 5 min to achieve full anesthesia.
   6. Once the mouse is fully anesthetized (i.e., slow but steady breathing rates at approximately one cycle per 2 s, coupled with the absence of a tail-pinch reflex), remove the mouse from the induction chamber and place the mouse head in the stereotaxic frame.
   7. Place the anesthesia tubing nose cone over the mouse's snout.
   8. Maintain anesthesia using 1.5% isoflurane, with carrier gas at a 1 L/min flow rate until the end of surgery. Periodically check the breath frequency, and deliver a tail or toe pinch at least every 15 min throughout surgery to assess appropriate anesthesia. Adjust the isoflurane concentration as appropriate to maintain the desired depth of anesthesia.
   9. Fill a microliter syringe (10 µL) with the virus solution. Then, fix the syringe onto the matched microinjector pump of the stereotaxic device.
3. Injection surgery
   1. Administer buprenorphine (1 mg/kg, subcutaneously) using a disposable insulin syringe and apply lubricant ophthalmic ointment to the mouse's eyes.
   2. Remove the hair from the scalp on top of the head by trimming with regular scissors 1.
   3. Clean the scalp skin with gauze and cotton tipped applicators. Disinfect the skin using 75% ethanol first and then betadine. Repeat this three times (i.e., ethanol followed by betadine), leaving the final application of betadine on the skin.
   4. Re-check the anesthetic depth to ensure that the mouse is fully anesthetized (i.e., slow but steady breathing rates at approximately one cycle per 2 s, coupled with the absence of a tail-pinch reflex). Make a ~1 cm incision using regular scissors 2 along the midline to expose the right parietal skull, and remove the periosteum using a cotton tipped applicator.
   5. Mark two points on the skull using a marker pen at these coordinates: point A: 2.5 mm posterior to the Bregma and 1 mm lateral to the midline over the right hemisphere; point B: 2.5 mm posterior to the Bregma and 2 mm lateral to the midline over the right hemisphere.
   6. Carefully drill two holes through the skull at the marked coordinates using an electric dental drill with a fine EF4 carbide bit.
      NOTE: Be careful not to damage the brain tissue.
   7. Adjust the stereotaxic frame to align the microliter syringe needle tip to the skull hole that is 1 mm lateral to the midline.
   8. Lower the syringe needle to touch the brain surface, and then set that location as the zero point (for the z-axis). Lower the needle tip into the brain cortex to a depth of 0.5 mm, and slowly infuse 1 µL of the virus solution at a speed of 200 nL/min.
   9. Wait 5 min after the virus solution is completely injected into the brain tissue before withdrawing the needle to prevent backflow of the solution.
   10. Slowly withdraw the syringe needle. Repeat the injection at the other site. Suture the incision with a sterile, non-absorbable surgical suture (6-0 gauge).
   11. Administer cefazolin [500 mg/kg (333 mg/mL, usually ~45 µL), intramuscularly], and meloxicam (5 mg/kg, subcutaneously) after surgery while the animal is still anesthetized.
   12. Release the mouse from the stereotaxic frame and discontinue anesthesia. Place the mouse in a clean cage above a heating blanket, and monitor the animal until it is ambulatory (approximately 15 min). Then, transfer to the home cage.
   13. Administer buprenorphine (1 mg/kg, subcutaneously) and meloxicam (5 mg/kg, subcutaneously) again at 8 h, 16 h, and 24 h, respectively, after the first buprenorphine injection.
       ​NOTE: At 2-4 weeks after virus injection, the mouse receives TBI and cranial window implantation at the same site as the AAV injection.

2. Administration of a repetitive TBI induction

NOTE: The TBI parameters are adjusted from previous reports^7,8, in which the TBI impact was delivered once. The protocol here applies the same parameter, except increasing the total impact number to 10.

1. TBI equipment
   1. Use a custom-built portable device for the administration of a closed-skull TBI (Figure 2A) to the mouse head on the right-side, as indicated in Figure 2B.
2. Preparation before surgery
   1. Autoclave surgery equipment, including regular scissors, a surgical caliper, surgical spring scissors, surgical forceps, mosquito forceps, headpost pieces, gauze, and cotton-tipped applicators.
   2. Weigh and record the mouse's body weight 2 h before surgery. To minimize brain edema during cranial window implantation, inject dexamethasone sodium phosphate at a dose of 4.8 mg/kg [2 mg/mL, usually ~70 µl (need to inject at two sites)] into the quadriceps muscle using an insulin syringe. After the injection of dexamethasone, but prior to starting TBI surgery, prepare the glass windows as indicated in step 3.1 for later use.
   3. Disinfect the surgical stage and TBI equipment using 75% ethanol, and expand the disposable sterile surgical drape to cover the surgery stage on the stereotaxic platform. Turn on the heating apparatus and the temperature monitor, setting the target temperature to 37 °C.
   4. Open the oxygen tank valve and adjust the oxygen flow to 1.5 L/min. Open the anesthesia machine and set the isoflurane level value to three.
      ​NOTE: 100% pure oxygen can help the animal to survive the TBI impacts.
   5. Put the mouse into the induction chamber and let it stay for 5 min to achieve full anesthesia.
   6. Once the mouse is fully anesthetized (i.e., slow but steady breathing rates at approximately one cycle per 2 s, coupled with the absence of a tail-pinch reflex), remove the mouse from the induction chamber and place the mouse head in the stereotaxic frame.
   7. Place the anesthesia tubing nose cone over the mouse's snout.
   8. Maintain anesthesia using 1.5% isoflurane mixed with 100% pure oxygen at a 1 L/min flow rate until the end of surgery. Periodically check the breath frequency, and deliver a tail or toe pinch at least every 15 min throughout surgery to assess appropriate anesthesia. Adjust isoflurane concentration as appropriate to maintain the desired depth of anesthesia.
3. TBI surgery
   1. Administer buprenorphine (1 mg/kg, subcutaneously) using disposable insulin syringes and apply ophthalmic ointment to the mouse's eyes.
   2. Remove the hair from the top of the head by trimming with scissors 1, and then applying depilatory agent for 1 min.
      ​CAUTION: Do not apply the depilatory agent product for more than 3 min on the scalp, as it is a skin irritant. Avoid getting any depilatory agent onto the mouse's eyes.
   3. Clean the scalp skin by applying gauze and cotton-tipped applicators. Then, disinfect the skin, using 75% ethanol first and then betadine. Repeat this three times (i.e., ethanol followed by betadine), leaving the final application of betadine on the skin.
   4. Re-check the anesthetic depth to ensure that the mouse is fully anesthetized (i.e., slow but steady breathing rates at approximately one cycle per 2 s, coupled with the absence of a tail-pinch reflex). Tent the skin with surgical forceps 3, and, make a 12-15 mm long midline incision starting approximately 3 mm posterior from the eyes.
   5. Excise the skin over the left and right hemisphere of the skull using spring scissors 2.
   6. Once the skull is exposed, remove the periosteum by gently rubbing with a sterile cotton-tipped applicator and flushing with sterile saline.
   7. Visually inspect the condition of the skull to ensure that it is intact, except for the two small holes, which were made for the previous virus injection surgery.
   8. Dry the skull area and mark the TBI impact site at the following coordinates: 2.5 mm posterior to the Bregma and 2 mm lateral from the sagittal suture to the right (Figure 2B).
   9. Quickly remove the mouse from the stereotaxic frame and place the head on the buffer cushion under the TBI device.
   10. Align the impactor tip with the marked impact site.
   11. Lift the metal column by pulling the tethered nylon string to 15 cm above the mouse head and then release it, allowing the weight to fall freely onto the transducer rod, which is in contact with the skull-top at the TBI site. Do not touch the mouse head when delivering the TBI impact.
   12. Move the mouse onto the heating blanket, and place it on its back while monitoring the breathing status.
   13. Once the mouse rights itself from a supine to a prone position, place the mouse into the isoflurane induction chamber for ~5 min with 3% isoflurane mixed with 100% pure oxygen at a 1.5 L/min flow rate.
   14. Once the mouse is fully sedated (i.e., slow but steady breathing rates at approximately one cycle per 2 s, coupled with the absence of a tail-pinch reflex), repeat steps 2.3.9-2.3.14 to achieve a total of 10 impacts.
       NOTE: Ten TBI impacts have been found to induce robust phenotype with low mouse mortality in our study (data not published yet). The parameters may be adjusted to achieve a different severity of brain injury by increasing or decreasing the number of impacts and/or the height from which the weight is released. All parameters are subject to approval by local IACUC.
   15. Check the skull under a surgical microscope, and remove the mouse from the study if a skull fracture has occurred.
   16. For a sham surgery, follow the same procedures as described above, including placement of the animal under the impactor, but without delivering the TBI impacts.
   17. After the mouse rights itself from a supine to a prone position following the 10^th TBI impact, place the mouse into the isoflurane induction chamber for ~5 min. Follow steps 2.2.6-2.2.8 to place the mouse head onto the stereotaxic frame for the cranial window implantation surgery described below.

3. Cranial window implantation surgery

NOTE: The cranial window implantation steps below were adopted from Goldey et al.⁹, and their specifications of the headpost and the imaging well were applied here.

1. Window preparation
   NOTE: Complete the window preparation in step 2.2.2 before starting the TBI surgery. The window is made from two round glass coverslips (one 3 mm and one 5 mm in diameter, as indicated by Figure 2C) that are joined together by transparent optical adhesive, as described below.
   1. Sanitize the glass coverslips by submerging them in 75% ethanol for 15 min. Take the glass coverslips out of the ethanol and leave them to dry on a sterile surface (i.e., the lid of a sterile 24-well plate) for ~10 min.
   2. Fill an insulin syringe with transparent optical glue while avoiding bubble formation. Under microscope 1 (Table of Materials) at 0.67x magnification, put a small drop (~1 µL) of optical glue at the center of the 5 mm coverslip. Then, immediately place the 3 mm coverslip on top, and center it with the 5 mm coverslip.
   3. Gently apply pressure using fine forceps to spread the glue evenly. Discard the glass window if a bubble or wall forms around the 3 mm cover slip.
   4. To cure the glue, place the coverslips into a UV box for 150 s at a power of 20 x 100 µJ/cm². Check that the two coverslips are securely bonded, by using forceps 4 to gently nudge the side of the 3 mm slip. If the coverslip moves, place it back in the UV box for an additional 60 s. Store the glass window in a sterile 24-well plate for later use.
2. Rough the skull surface.
   NOTE: From here on, carry out all steps in section 3 under a surgical microscope. Start with 10x, and adjust to the desired magnification.
   1. Gently and slowly drill the surface of the skull using a FG4 carbide bit at a low rotor speed (i.e., output number set to ~1-2) to remove the remaining periosteum and create a rough skull surface, such that the dental cement binds securely with the skull. A scalpel can also be used here instead of a drill as an alternative.
   2. Use saline to flush and clear bone dust from the skull surface.
3. Separate the muscles.
   NOTE: Muscle separation serves to increase the surface area of the exposed skull bone that will serve as a contact point for the dental cement, thereby ensuring structural integrity of the implant. This muscle separation step usually exposes ~3 mm of the temporal skull plate.
   1. At approximately 5 mm posterior to the eye, where the suture connecting the parietal and temporal skull is located, gently insert the closed fine tips (#5/45 forceps) to separate the lateral muscles from the skull, and gently move the closed tips in the posterior direction until the Lambdoid suture.
   2. Separate the lateral muscles on the side where implantation of the cranial window will occur.
      NOTE: Be careful not to separate the muscles too close to the eye, to avoid injuring the ophthalmic artery; otherwise, severe and persistent bleeding may occur. Do not separate muscles from the occipital bone, as the mouse requires these muscles to raise its head.
   3. Wash away the debris from the surgical site using saline and dry the area with gauze. Gel foam can be applied to the surgical site to stop the bleeding.
4. Implant the headpost.
   1. Use a custom-made titanium headpost (Figure 2D), described in a previous publication⁹, to fix the mouse head securely while performing the craniotomy surgery, and for subsequent two-photon imaging.
   2. Use a marker pen and a surgical caliper to trace the circumference of the craniotomy on the clean and dry skull on the right side. The center point of the traced circle is 2.5 mm posterior to the Bregma and 1.5 mm off the sagittal suture on the right hemisphere. The diameter of the traced circle is about 3.2-3.5 mm, slightly larger than the 3 mm glass coverslip. Ensure the craniotomy circle can cover the virus injection sites (the two holes made while injecting the virus can be used as a reference) and the TBI site.
   3. Loosen the ear bar and rotate the head so that the craniotomy plane is perfectly horizontal, and then tighten the ear bar again.
   4. Use the wood stick of a cotton tipped applicator to add two small drops of superglue to the front and back edge of the headpost.
   5. Position the titanium headpost over the center of the craniotomy, and quickly adjust it to rest within the same plane as where the cranial window will be implanted. Apply light pressure until the superglue is dried; this usually takes ~30 s.
   6. Prepare dental cement in a pre-cooled ceramic mixing dish (staying at least 10 min in a -20 °C freezer): combine 300 mg of cement powder, six drops of quick base liquid, and one drop of catalyst, and then stir the mixture until thoroughly mixed (~15 times).
      NOTE: The cement needs to be pasty. If it is too thin, stir in a little more cement powder. If it is too thick, stir in quick base liquid one drop at a time until a pasty consistency is achieved.
   7. Quickly apply a generous amount of dental cement mixture to the outside perimeter of the traced circumference, and cover any exposed bone surface. However, do not cover the site of the craniotomy. Allow ~15 min for the dental cement to dry and harden before proceeding.
   8. Release the ear bar and secure the headpost to the metal frame to ensure that the head is stable for precise drilling along the marked craniotomy circumference. If there is cement over the craniotomy site, use the FG4 carbide bit to drill and remove it.
5. Craniotomy
   1. Use a surgical caliper to verify the diameter of the marked circle, as defined in step 3.4.2. Adjust as necessary, so that the cranial window will fit snugly inside the craniotomy.
   2. Using an electric dental drill, etch and thin the skull along the outside of the marked circle, using a FG4 carbide bit first (speed set to an of output ~9-10). This creates a "track" within which to thin the skull.
      CAUTION: To minimize heat injury and achieve a smooth track, keep moving the drill bit. Do not drill the same place for more than 2 s.
   3. Periodically, stop drilling and irrigate the whole area with sterile saline, to reduce heating from the drill and to wash away the bone dust.
   4. Continue to thin the skull using an FG1/4 carbide bit, as described in step 3.5.2, until the skull is paper-thin and transparent.
      NOTE: Using two hands to hold the drill can make it easier to control the drill and avoid inserting the drill bit into the brain.
   5. Complete the skull thinning using an EF4 carbide bit. When a crack occurs between the bone flap and the surrounding skull bone, sometimes there is a release of cerebral spinal fluid (CSF), indicating that the skull has been completely drilled through.
   6. Continue thinning and drilling through the rest of the skull along the track. Avoid drilling through the point where an obvious vasculature crosses under the skull to prevent bleeding.
   7. Insert a fine forceps tip (forceps 1) ~0.5 mm through the cracked place, and lift the bone flap gently upward without indenting the underlying brain.
   8. After the bone flap is removed, irrigate the craniotomy area with saline. The brain surface could be 1-2 mm higher than the craniotomy edge.
   9. Starting from this step, always cover the exposed brain with saline to protect the brain tissue.
   10. Bleeding may occur when lifting the bone flap at the original AAV injection sites due to the tissue adhesion and vasculature growth after the injection surgery. If this happens, gently press the site with a dry cotton tipped applicator for ~2 min (or longer as needed). Gel foam can be used to stop the bleeding. Do not use chemicals or heating forceps to stop the bleeding, as this can cause injury.
   11. Use the forceps 1 to gently remove the visible arachnoid matter.
6. Implant glass window
   1. Use the surgical forceps 2 to pick up the sterile glass window, with the 3 mm glass coverslip facing down. Place and adjust the glass window above the craniotomy site to make sure the window can fit snugly to the craniotomy edge. The 5 mm glass coverslip is on top.
   2. Prepare dental cement as follows: combine 100 mg of cement powder, two drops of quick base liquid, and one drop of catalyst. Stir the mixture until thoroughly mixed (~15 times). Wait ~6 min until the cement becomes pasty and thick. If the cement is too thin, it may seep into the space under the window in step 3.6.4 and obscure the window.
   3. While waiting for the cement to become pasty and thick, apply an adequate amount of pressure to the window through a stereotaxic manipulator, to check that the skull can securely and tightly contact the glass window. Ensure that the dental cement liquid in step 3.6.4 will not reach the space underneath the glass and thus obscure the window.
   4. Use an adjustable precision applicator brush to add a small amount of cement alongside the window edge to seal the glass window with the skull. Wait for ~10 min to let the cement completely dry, and then gently release and remove the manipulator above the window. As of this step, it takes ~4 h to finish the 10 TBI impacts and implant the headpost and cranial window.
   5. Trim the dental cement using the dental drill with an FG4 carbide bit if there is excess cement covering the window.
      NOTE: Excessive cement around the window may prevent the two-photon objective lenses from approaching the window surface.
7. Implanting the imaging well
   NOTE: An imaging well (Figure 2D) is a rubber ring with an outside diameter of ~1.6 cm, that matches the headpost top surface and holds water above the cranial window for two-photon imaging.
   1. After the window implantation is completed, drill away the dental cement debris on the headpost top surface, and clean the area using wet surgical gauze. Allow the area to dry for ~3 min.
   2. Use a cotton tipped applicator to dip a small amount of superglue and paste it on the headpost top surface. Quickly place the rubber ring onto the headpost. Apply medium pressure on the rubber ring for ~2 min to ensure close contact with the headpost. Use superglue sparingly, otherwise, it can adhere to the glass window and obscure the two-photon imaging.
8. Analgesic and antibiotic administration
   1. Immediately after implanting the imaging well, but prior to discontinuing isoflurane, administer cefazolin [500 mg/kg (333 mg/mL, usually ~45 µL), intramuscularly], and meloxicam (5 mg/kg, subcutaneously) using disposable insulin syringes.
   2. After drug administration, discontinue the anesthesia, release the mouse from the stereotaxic frame, and return the mouse to its home cage, which is above a heating blanket.
   3. Closely monitor the mouse for ~15 min until it is ambulatory.
      NOTE: House the mice individually, as they may bite the rubber imaging well of other mice. Provide food and water gel close to the mouse in the cage for access ad libitum.
   4. Administer buprenorphine (1 mg/kg, subcutaneously) and meloxicam (5 mg/kg, subcutaneously) again every 8 h after the previous dose until 48 h after the cranial window implantation surgery.

4. Intravital two-photon imaging

1. Use microscope 2 (Table of Materials), equipped with a tunable coherent multiphoton laser and a 20x magnification objective (NA 1.0; water-immersion), for intravital imaging¹⁸. The filter used for the EGFP signal is "BP 500-550".
2. Prepare the cranial window mouse for intravital imaging.
   1. Starting from the surgery day (designated as day 0), carry out two-photon imaging at designed time points post-TBI. Place the cranial window mouse into the anesthesia induction chamber, and administer 3% isoflurane mixed with carrier gas at a 1.5 L/min flow rate for 5 min.
      NOTE: The carrier gas can be either room air or 100% oxygen.
   2. Once the mouse is fully anesthetized (i.e., slow but steady breathing rates at approximately one cycle per 2 s, coupled with the absence of a tail-pinch reflex), remove the mouse from the induction chamber and quickly clamp the headpost to a bracket. Let the mouse's torso lay on a round plastic plate (19 cm diameter) that is equipped with the bracket.
      NOTE: A hand-warm pad was placed under the mouse's torso to provide heat support during intravital imaging.
   3. Apply lubricant ophthalmic ointment onto the mouse's eyes and place the anesthesia tubing nose cone over the mouse's snout. Maintain anesthesia by using 1.5% isoflurane with carrier gas at a 1 L/min flow rate.
   4. Periodically check the breath frequency, and deliver a tail or toe pinch at least every 15 min throughout imaging to assess appropriate anesthesia. Adjust isoflurane concentration as appropriate to maintain the desired depth of anesthesia.
   5. Align the mouse head to ensure that the cranial window is directly underneath the two-photon objective lens. Add some water into the imaging well above the cranial window. Lower the objective so that it is immersed in the water.
3. Intravital imaging of intracranial window mice
   1. Turn on the scope mercury lamp. View the brain with epifluorescence through the ocular first.
      NOTE: The vasculature appears black. Select an area where the window is clear. Turn off the epifluorescence, set the laser wavelength to 860 nm for the EGFP signal, and adjust the laser setting for optimal (i.e., bright but not saturated) signal.
   2. Set the scan mode to Frame and the line step to 1. Set the averaging number to 16, the bit depth to 8-bit, the mode as Line, and the method as Mean.
   3. Use the vasculature pattern as a "reference map" to image the same brain region for subsequent longitudinal imaging. Image the superficial level (layer I, less than 100 µm from the meningeal surface) for three planes where the cortical vasculature dominates through a z-stack mode, with an interplane interval of 10 µm, as indicated in Figure 3A.
   4. Image six planes at deep level (layer IV and V, ~400 µm deeper than the superficial level), through a z-stack mode as indicated in Figure 3A, with an interplane interval of 10 µm and a scanning speed of 8.
   5. After finishing the imaging (~20 min per mouse), discontinue the anesthesia, release the mouse from the frames, and return it to its home cage that is above a heating blanket. Monitor the mouse consecutively until it is ambulatory, which usually takes ~7 min.
   6. Longitudinally image the mouse at day 0, 1 week, and 4 months post-TBI surgery.

Representative Results

As proof of concept for this protocol, viral particles expressing AAV-Syn1-EGFP were injected into the brain cortex of male TDP-43^Q331K/Q331K mice (C57BL/6J background)¹⁹ at the age of 3 months. It is noted that wild-type C57BL/6J animals can also be used, however this study was carried out in TDP-43^Q331K/Q331K mice because the laboratory is focused on neurodegenerative disease research. A TBI surgery was performed 4 weeks after AAV injection. Within the same surgical setting, the headpost and cranial window were implanted. The mouse was serially imaged using a two-photon microscope at day 0, 1 week, and 4 months post-TBI surgery (Figure 1). In general, the injection surgery took ~30 min, and the TBI surgery took ~1 h per mouse. During the TBI surgery, the mice generally required a longer amount of time to right themselves from a supine position to a prone position after subsequent impacts, compared to the initial impacts. For example, a mouse might have required 2 min to right its position after the first impact, but required 10 min to right its position after the 10^th impact. For cranial window implantation surgery, ~3 h was usually required to perform all the steps, but took longer than necessary if persistent bleeding occurred. Two-photon imaging usually required 20 min per mouse to acquire all the images. For the current study involving three mice with the expression of EGFP, the animals did not show evidence of a skull fracture, nor did they die during surgery or up until 4 months after surgery. Both the morbidity and skull fracture rate were 0%. This is consistent with the relatively low (<10%) mortality rate in a similar model of inducing TBI but without a cranial window implant^7,8.

A weight-drop device (Figure 2A), that has been previously reported^7,8, was used to deliver TBI impacts onto the mouse head on the right-side, as indicated in Figure 2B. A transparent plastic tube (no. 5 in Figure 2A; 60 cm in length and 1.4 cm in inner diameter) was securely and vertically clamped to a metal bracket, and a plastic impactor column (no. 7 in Figure 2A; 10 cm in length and 1.3 cm in diameter, with a flat round tip of 2 mm in diameter) was placed in the vertical tube. A 50 g metal weight (no. 6 in Figure 2A) was placed above the impactor and tethered with a nylon string (no. 4 in Figure 2A). A buffer cushion (no. 8 in Figure 2A; 9 cm x 9 cm x 1 cm [length x width x depth], made of polyethylene foam and cotton gauze) was placed under the mouse head to buffer and absorb the impact energy.

The cranial window was made of two glass coverslips combined by optical adhesive (Figure 2C), and the glass coverslip of 3 mm diameter was the side touching the brain surface. Two-photon imaging was carried out at two different depths (Figure 3A) from the brain surface: 1) a superficial level where the cortical vasculature was abundant and had a relatively large lumen diameter that appeared black, but with sparse EGFP positive cell bodies; and 2) a deeper level where the vasculature was sparse and had a small lumen diameter, with many EGFP positive cells visible.

At ~4 h (day 0) after surgery, two-photon imaging was carried out at three planes, with an interval of 10 µm at the superficial level (layer I, less than 100 µm from the meningeal surface) first where the vasculature appeared black (indicated by the dashed red lines in Figure 3B), and was used as a reference map for the next imaging session to locate the original imaging region. At this superficial level, the cortical vasculature and neuronal processes were the predominate structures that could be observed, whereas the EGFP positive cell bodies were sparse. After imaging within the superficial level, the focus was adjusted downward by ~400 µm, deeper than the superficial level, into the cortex (layer IV and V) to image the EGFP positive neuronal cell bodies. Images were acquired within six planes, with an interval of 10 µm. EGFP protein expression was diffusely distributed throughout the cell body, as indicated in Figure 3C. There was occasional occurrence of some EGFP puncta outside the cell bodies, which could represent EGFP inclusions that formed over time within axons or dendrites. This protocol results in AAV-SYN1-EGFP expression ~2-3 mm surrounding the injection site, which can be defined by histological analysis of post-mortem tissues.

At 1 week and 4 months post-TBI, the vasculature pattern observed at the day 0 time point was used as a reference to locate the same imaging region (Figure 3D,F). The vasculature pattern in Figure 3D,F is similar to that in Figure 3B. The imaging procedure for the 1 week and 4 months post-TBI was the same as that described above for the day 0 time point, and EGFP expression was detected throughout the cell body as before (Figure 3E,G). The fluorescence intensity at day 0 and 4 months were similar at both the superficial and deeper levels. However, the fluorescence intensity at 1 week was lower than that of day 0 and 4 months at both the superficial and deep levels, which could be due to the translational repression reported for other TBI models²⁰. Notably, the quality of the images at 4 months compared to the day 0 time point demonstrates that the cranial window has maintained clarity and integrity, and that the viral expression is still robust 4 months post-surgery for effective intravital imaging. As EGFP expresses as a relatively diffuse protein, the fluorescence signals may be better resolved and discreet when EGFP is fused to a protein of interest.

Figure 1: The timeline for this intravital imaging protocol. The protocol is initiated with virus injection. TBI and cranial window surgery are performed within the same surgery session at 2-4 weeks after virus injection. Intravital imaging is carried out at day 0, 1 week, 4 months after TBI. Please click here to view a larger version of this figure.

Figure 2: Diagrams for the TBI device, TBI impact site, and window preparation. (A) Equipment for the weight-drop TBI device. 1: pedestal, 2: bracket, 3: adjustable clamp, 4: nylon string, 5: weight dropping tunnel, 6: metal weight column (50 g), 7: impactor, 8: buffer cushion. (B) A schematic of the virus injection and TBI impact site, with the coordinates 2.5 mm posterior to the Bregma and 2 mm lateral from the sagittal suture to the right. (C) A schematic for preparing the glass window. Two glass cover slips, 3 mm and 5 mm in diameter, are combined using optical adhesive. (D) Pictures of the metal headpost and imaging well. Please click here to view a larger version of this figure.

Figure 3: Schematic of imaging planes and the image data. (A) Multiple planes are imaged at a superficial level where the vasculature is located and a deep level. The images are collected as follows: three planes at the superficial level, where the cortical vasculature and neuronal processes are the predominate EGFP-positive structures, and six planes at the deep level, where EGFP-positive cell bodies are predominately observed, with a 10 µm interval between the neighboring planes. Each plane is of a 425.10 µm x 425.10 µm size. (B) Representative image of a plane in the superficial level at the day 0 time point. The dashed red lines indicate the vasculature outline that can be used as a reference to locate the original imaging place for subsequent time points. (C) Representative image of a plane in the deep level at the day 0 time point soon after TBI. (D) Representative image of a plane in the superficial level at the 1 week time point. The dashed red lines denote the vasculature, similar to the outline on day 0 in Figure 3B, confirming that the two-photon imaging was carried out at a similar location on day 0 and 1 week after TBI. (E) Representative image in the deep level at the 1 week time point post-TBI. (F) Same as B and D, at 4 months post-TBI. (G) Same as C and E, at 4 months post-TBI. Scale bar: 50 µm. Please click here to view a larger version of this figure.

Discussion

In this study, AAV injection, TBI administration, and a headpost with cranial window implantation were combined for longitudinal imaging analysis of EGFP-labeled neurons within the mouse brain cortex (layers IV and V) to observe the effects of TBI on cortical neurons. This study notes that the TBI site chosen here, above the hippocampus, provides a relatively flat and broad surface for implantation of the cranial window. Conversely, the skull is relatively narrow anterior to this site, and therefore it is difficult to ensure that the headpost will effectively contact the surface of the skull. While only the TDP-43^Q331K/Q331K mouse model was used in this study, considering the laboratory's research focusing on ALS and FTD¹⁹, this protocol should be applicable to most other mouse strains. In addition to labeling neurons as described here, various strategies can be used to label other cell types for intravital imaging. One approach is to express the genetically encoded fluorescent proteins in a cell-specific manner, using the Cre-Lox recombination system in genetically modified mice²¹. Another approach is to employ certain viral serotypes for cell-specific transduction of genetically encoded fluorescent proteins. Site-specific delivery can be achieved by performing the intracranial injection at the desired location in the brain. The efficiency and ease with which one can express cell-specific proteins via viral transduction for intravital imaging is an advantage over creating transgenic mouse models.

For the surgery protocols, there are multiple critical steps that need to be carefully performed. When drilling holes on the skull for virus injection at the start of the protocol, one needs to be cautious of damaging the tissue underneath the skull. If tissue damage occurs while drilling on the skull, there will be inflammation, tissue adhesion, and angiogenesis around the injury site, thereby increasing the risk of bleeding during the craniotomy for cranial window implantation. For administration of the TBI with the weight-drop device, the present study placed a cushion under the mouse head to buffer the TBI impact force, and thereby decrease the chance of a skull fracture. No obvious head motion on rotational directions and acceleration were observed, supporting the notion that this TBI model has a relatively low chance of diffusion axonal injury. Interestingly, Foda et al. used a similar weight-drop device to construct a closed-skull TBI injury on rats, and a larger, soft foam cushion (12 cm thick) was placed under the rat head so that it could easily move in response to the TBI impact force on rotational direction with acceleration, thereby resulting in diffusion axonal injury²². One of the advantages of the tube-guiding weight drop TBI model is that the severity of the trauma can be modulated by changing the weight drop height (i.e., a higher height for a larger force) and/or repeating the number of times the impact is delivered. For example, Flierl et al. used a similar weight-drop device as the present study to induce TBI on mice; the weight of the metal was 333 g, which induced a mild TBI when dropped from a height of 2 cm and a severe TBI when dropped from a height of 3 cm²³. The craniotomy step prior to window implantation also needs to be performed carefully, to avoid damaging the brain tissue under the skull. Periodically moving the drill bit to a different region on the skull helps to avoid over-drilling at the same spot, which can result in excessive heating, tissue damage, and bleeding; further, frequently irrigating with saline can also cool the drilling site. When implanting the glass window, it is important to align the glass plane such that it is parallel to the skull surface plane; a snug window fit within the skull prevents leakage of the dental cement liquid into the space between the window and the brain.

If the window is blurry at day 0 post-TBI surgery, but fluorescent signals are detected, the dental cement may have entered the space between the window and the brain, thereby forming a cement layer that covers the brain surface and obscures the fluorescence emission. In this situation, one can remove the window and the dental cement using the drilling method described in protocol step 3.5, and then implant a new glass window at the original site, as described in detail by Goldey et al.⁹. If the window becomes blurry at a subsequent stage during the longitudinal imaging process, one can attempt to remove potential debris by gently rubbing the window with a cotton-tipped applicator. If this does not resolve the issue, it may be due to regrowth of the bone and the meninges underneath the glass window. In this case, one can remove the window and drill to remove the regrown bone and/or tweeze apart and remove the meninges, followed by implantation of a new glass window at the original site, as described by Goldey et al.⁹. As shown in the results, EGFP expression and fluorescence is robust over a time course of ~4 months post-TBI. One can expect a decrease of fluorescence signal within the first week post-TBI, which is likely due to TBI-induced translational repression that causes a temporary reduction in global protein synthesis²⁰.

To maintain high imaging quality and achieve imaging in multiple planes, mice were under isoflurane anesthesia to limit movement. However, it is important to note that anesthesia may affect the study results. For example, it has been reported that mice under isoflurane anesthesia exhibit different microglial activity in response to photodamage compared to awake mice²⁴. For two-photon imaging, the scanning speed and the number of scans for signal averaging (set as "number", under "averaging") are the main factors that can determine image resolution. To optimize resolution, one can reduce the scanning speed and increase the averaging number, however, a slower speed and higher averaging number increases the imaging time for a particular field of view and may cause photo bleaching. The present study aims to accomplish chronic long-term imaging on the same animal at the same brain location. To avoid potential photo bleaching while achieving sufficient resolution, two-photon imaging was carried out at a scanning speed of 8 with an averaging number of 16.

An alternative approach to performing a craniotomy and implanting a glass window is to thin the skull, to the extent that it is transparent and "paper thin" for live imaging^25,26,27. The thin-skull window can maintain the intactness of the skull, and thus avoid the inflammation induced by the surgical operation. Therefore, the thin-skull window approach may be preferred for live imaging with inflammation-relevant markers and/or over a relatively shorter period of time (i.e., ~14 days post-surgery, as reported²⁵). For long-term imaging (i.e., 4 months, as described here) of chronic neurodegenerative processes, in addition to assessing the acute effects of TBI on the same animal, it is recommended to use a glass window implanted by a craniotomy method.

As mentioned in the introduction, there are several notable advantages of intravital imaging for studying various biological and pathologic events in the mammalian brain. However, there are some limitations of this protocol as well. For example, contrecoup brain injury describes the brain contusion or hematoma remote from, usually opposite to, the force contacting site^28,29,30. In the present study, the TBI impacts were delivered to the parietal lobe; contrecoup injury would be expected ventrally and inaccessible to intravital imaging. A histological analysis could be used as a complementary approach to further examine phenotypes of interest outside the impact site covered by the cranial window. In addition, exogenous overexpression of certain proteins may also induce toxicity or pathology. In the present study, some occasional EGFP puncta were observed, which may represent accumulations due to protein overexpression. Therefore, it is recommended to include a negative-control protein (e.g., EGFP or RFP alone) in the study design to address this possibility, particularly if the protein of interest is prone to forming punctate structures. The number of proteins that one can study simultaneously is limited by the lasers and capabilities of the two-photon system. It may be possible to image more than one fluorophore (e.g., EGFP, RFP, etc.) by two-photon microscopy, although multiplexing capabilities with conventional wide-field and confocal microscopes often allow analyses of more proteins within a single tissue sample. Finally, it is important to include a sham control that receives all the same procedures as the TBI animal, except the weight-drop impacts. Cranial window implantation surgery is an invasive operation and can cause some local changes, such as inflammation and altered intracranial pressure, which could affect the TBI process. A sham control helps the experimentalist assess phenotypes that are due to the impact versus the surgical procedures.

In summary, this protocol introduces a method to longitudinally image proteins specifically in the neurons of the mouse brain cortex in response to TBI. This protocol can be modified to analyze the expression and localization of various cell-specific proteins in response to TBI. The goal of this protocol is to provide an approach for studying the immediate and long-term consequences of TBI in the mammalian brain.

Materials

Name	Company	Catalog Number	Comments
Adjustable Precision Applicator Brushes	Parkell	S379
BD insulin syringe	BD	NDC/HRI#08290-3284-38	5/16" x 31G
Betadine	Purdue	NDC67618-151-17	including 7.5% povidone iodine
Buprenorphine	PAR Pharmaceutical	NDC 42023-179-05
Cefazolin	HIKMA Pharmaceutical	NDC 0143-9924-90
Ceramic Mixing Dish	Parkell	SKU: S387	For dental cement preparation
Cotton Tipped Applicators	ZORO	catlog #: G9531702
Catalyst	Parkell	S371	full name: "C" Universal TBB Catalyst
Dental cement powder	Parkell	S396	Radiopaque L-Powder for C&B Metabond
Dental drill	Foredom	H.MH-130
Dental drill controller	Foredom	HP4-310
Dexamethasone	Phoenix	NDC 57319-519-05
EF4 carbide bit	Microcopy	Lot# C150113	Head Dia/Lgth/mm 1.0/4.2
Ethonal	Fisher Scientific	04355223EA	75%
FG1/4 carbide bit	Microcopy	Lot# C150413	Head Dia/Lgth/mm 0.5/0.4
FG4 carbide bit	Microcopy	Lot# C150309	Head Dia/Lgth/mm 1.4/1.1
Headpost	N/A	N/A	Custom-manufactured
Heating apparatus	CWE	TC-1000 Mouse	equiped with the stereotaxic instrument and be used while operating surgery
Heating blanket	CVS pharmacy	E12107	extra heating device and be used after surgery
Isoflurane	Pivetal	NDC 46066-755-03
Isoflurane induction chamber	Vetequip	89012-688	induction chamber for short
Isoflurane volatilizing machine	Vetequip	911103
Isoflurane volatilizing machine holder	Vetequip	901801
Leica surgical microscope	Leica	LEICA 10450243
Lubricant ophthalmic ointment	Picetal	NDC 46066-753-55
Marker pen	Delasco	SMP-BK
Meloxicam	Norbrook	NDC 55529-040-10
Microinjection pump and its controller	World Precision Instruments	micro4 and UMP3
Microliter syringe	Hamilton	Hamilton 80014	1701 RN, 10 μL gauge for syringe and 32 gauge for needle, 2 in, point style 3
Mosquito forceps	CAROLINA	Item #:625314	Stainless Steel, Curved, 5 in
Depilatory agent	McKesson Corporation	N/A	Nair Hair Aloe & Lanolin Hair Removal Lotion
Microscope 1	Nikon	SMZ745	Nikon microscope for cranial window preparation
Microscope 2	Zeiss	LSM 7 MP	two-photon microscope
Multiphoton laser	Coherent	Chameleon Ultra II, Model: MRU X1, VERDI 18W	laser for two-photon microscopy
Non-absorbable surgical suture	Harvard Apparatus	catlog# 59-6860	6-0, with round needle
Norland Optical Adhesive 81	Norland Products	NOA 81
No-Snag Needle Holder	CAROLINA	Item #: 567912
Quick base liquid	Parkell	S398	"B" Quick Base For C&B Metabond
Regular scissor 1	Eurostat	eurostat es5-300
Regular scissor 2	World Precision Instruments	No. 501759-G
Round cover glass 1	Warner instruments	CS-5R Cat# 64-0700	for 5 mm of diameter
Round cover glass 2	Warner instruments	CS-3R Cat# 64-0720	for 3 mm of diameter
Rubber rings	Orings-Online	Item # OO-014-70-50	O-Rings
Saline	Bioworld	L19102411PR
Spring scissor 1	World Precision Instruments	No. 91500-09	tip straight
Spring scissor 2	World Precision Instruments	No. 91501-09	tip curved
Stereotaxic platform	KOPF	Model 900LS
Super glue	Henkel	Item #: 1647358
surgical Caliper	World Precision Instruments	No. 501200
Surgical forceps 1	ELECTRON MICROSCOPY SCIENCES	Catlog# 0508-5/45-PO	style 5/45, curved
Surgical forceps 2	ELECTRON MICROSCOPY SCIENCES	catlog# 0103-5-PO	style 5, straight
Surgical forceps 3	ELECTRON MICROSCOPY SCIENCES	catlog# 72912
Surgical forceps 4	ELECTRON MICROSCOPY SCIENCES	Catlog# 0508-5/45-PO	style 5/45, curved
Surgical gauze	ZORO	catlog #: G0593801
Surgical lamp	Leica	Leica KL300 LED
UV box	Spectrolinker	XL-1000	also called UV crosslinker
Vaporguard	Vetequip	931401
Vetbond Tissue Adhesive	3M Animal Care	Part Number:014006