modeling neonatal intraventricular hemorrhage through intraventricular injection of hemoglobin

Modeling Neonatal Intraventricular Hemorrhage Through Intraventricular Injection of Hemoglobin

To begin, place the anesthetized rat prone in the stereotactic apparatus, with its nose positioned in the anesthesia adapter. Then, secure the head by tightening the nonrupture ear bars on the external auditory meatus.
To clean the head, first, touch a sterile cotton-tipped applicator soaked in betadine at the center of the head and move outwards to spread the betadine in circles. Then, repeat the process with an applicator soaked in 70% ethanol.
Next, using a scalpel, make a 0.3-centimeter incision vertically down the center of the head, to expose the bregma of the skull. Then, dry the area using a cotton-tipped applicator.
To set up the stereotactic injector, draw the previously prepared hemoglobin solution into a 0.3 or 0.5-milliliter syringe, and place the syringe in the stereotactic injector system.
Next, turn on the stereotactic injector interface, and click on the configuration button to enter the injection volume and rate settings. Click on volume and set the volume at 20,000 nanoliters.
Then, click on infusion rate and set the rate at 8000 nanoliters per minute. Click on the reset position button to exit the configuration. Flush the needle tip by clicking the infuse button, until a small bead of hemoglobin appears at the needle tip.
Then use a cotton-tipped applicator to gently remove the bead. To begin the animal injection, set the bregma as zero on the stereotactic injecting system by adjusting the mediolateral and anteroposterior positions of the syringe.
Then, lower the syringe needle to touch the skull at the bregma gently. After identifying the coordinates of choice, raise the syringe needle one centimeter above the skull to clear the scalp.
Set the mediolateral and anteroposterior coordinates. Then, lower the syringe for the needle to gently touch the skull, and set the dorsoventral coordinate over 30 seconds.
Once the coordinates have been set, begin injection by clicking the RUN button on the stereotactic injector interface. After the injection is finished, leave the needle in place for two minutes to minimize the backflow of the solution.
Next, slowly withdraw the syringe along the dorsoventral coordinate over two minutes until the needle tip is two centimeters above the scalp. Then, rotate the stereotactic injector arm away from the operative field and close the scalp with a 6-0 monofilament suture.

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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Preparation of hemoglobin and CSF solutions
<ol>
	<li>Prepare a sterile artificial cerebrospinal fluid (aCSF) solution by adding 500 &#181;L of the aCSF solution to a 1.5 mL microtube and store on ice.</li>
	<li>Prepare a sterile 150 mg/mL hemoglobin solution by adding 75 mg of hemoglobin to 500 &#181;L of aCSF in a 1.5 mL microtube and store on ice.</li>
</ol>
2. Preparation of the animal for injection
<ol>
	<li>Turn the heating pad to the medium setting to maintain the body temperature of the rat.</li>
	<li>Anesthetize postnatal day 4 (P4) rats in an induction chamber filled with 3% isoflurane. 
	NOTE: Confirm sufficient anesthesia using the toe/tail-pinch response every 15 min. Monitor anesthesia with visual observation of tissue color, body temperature, and respiratory rate.</li>
	<li>Administer pain relief with a 5 mg/kg subcutaneous carprofen injection to the anesthetized rat.</li>
	<li>Place the anesthetized rat prone in the stereotactic apparatus with the nose positioned in the anesthesia adaptor with a constant flow of 1.5% isoflurane.</li>
	<li>Tighten non-rupture ear bars on the external auditory meatus to secure the head. 
	NOTE: Apply vet ointment to keep the eyes moisturized if the eyes are open at the age of injection.</li>
	<li>Clean the head, alternating with sterile cotton-tipped applicators soaked in betadine and 70% ethanol.
	<ol>
		<li>Touch the betadine-soaked applicator to the center of the scalp and spread the betadine in circles, moving outward.</li>
		<li>Repeat step 2.6.1.1 with the ethanol-soaked applicator.</li>
		<li>Repeat step 2.6.1.1 and step 2.6.1.2 3x.</li>
	</ol>
	</li>
	<li>Apply a sterile surgical drape to protect the surgical field.</li>
	<li>Using a sterile scalpel, make a 0.3 cm incision vertically down the center of the head to expose the bregma of the skull. 
	NOTE: If injecting from the lambda, expose the lambda of the skull instead of the bregma.</li>
	<li>Use a sterile cotton-tipped applicator to dry the area.</li>
</ol>
3. Setting up the stereotactic injector
<ol>
	<li>Draw the hemoglobin solution prepared in step 1.2 into a 0.3 mL sterile syringe with a 30G needle and place the syringe into the stereotactic injector system. 
	&#8203;NOTE: If generating control conditions, draw aCSF solution prepared in step 1.1 into a 0.3 mL sterile syringe and proceed with the protocol.</li>
	<li>Turn on the stereotactic injector interface and click on the Configuration button to input the injection volume and rate settings.
	<ol>
		<li>Click on Volume and set the volume at 20,000 nL (20 &#181;L).</li>
		<li>Click on Infusion rate and set the rate at 8,000 nL/min (8 &#181;L/min).</li>
	</ol>
	</li>
	<li>Exit Configuration by clicking on the Reset Pos button.</li>
	<li>Flush the needle tip by clicking on the Infuse button until a small bead of hemoglobin solution emerges at the needle tip.</li>
	<li>Gently wick the hemoglobin solution from the needle tip with a sterile cotton-tipped applicator.</li>
</ol>
4. Animal injection
<ol>
	<li>Set the bregma as zero on the stereotactic injector system by adjusting the mediolateral and anteroposterior positions of the syringe before lowering the tip of the flushed syringe needle to gently touch the skull at the bregma. 
	NOTE: If injecting from the lambda, set lambda as zero.</li>
	<li>Identify the coordinates of choice.
	<ol>
		<li>If injecting from the bregma, in P4 rats described here, use 1.5 mm lateral, 0.4 mm anterior, and 2.0 mm deep from bregma.</li>
		<li>If injecting from the lambda, use the following coordinates for P4 rats: 1.1 mm lateral, 4.6 mm anterior, and 3.3 mm deep from lambda.</li>
	</ol>
	</li>
	<li>Raise the syringe needle 1 cm above the skull to clear the scalp. When the syringe is raised, proceed to set the mediolateral and anteroposterior coordinates.</li>
	<li>Lower the syringe needle to gently touch the skull. Check that the needle is touching the skull.</li>
	<li>Set the dorsoventral coordinate over a 30 s period. 
	NOTE: While setting the dorsoventral coordinate, the needle will puncture the skull. Care must be taken to assure that the syringe passes through the skull without deforming the skull. Skull deformation is avoided by slowly withdrawing the needle along the dorsoventral coordinate if deformation occurs, then placing the needle back along the same trajectory. This allows the needle to pass through the hole in the skull with less force and no deformation.</li>
	<li>On the stereotactic injector interface, click on the Run button to begin injection.</li>
	<li>After the injection is finished, leave the syringe needle in place for 2 min to minimize backflow of the solution.</li>
	<li>Withdraw the syringe slowly along the dorsoventral coordinate over 2 min until the needle tip is 2 cm above the scalp.</li>
	<li>Rotate the stereotactic injector arm away from the operative field.</li>
</ol>
5. Postoperative care
<ol>
	<li>Close the scalp with a 6-0 monofilament suture. Make one simple interrupted suture at the center of the 0.3 cm incision.</li>
	<li>Remove the pup from anesthesia and place it onto a secure area on the heating pad.</li>
	<li>Return the rodent to the home cage to recover from the anesthesia under the care of its dam. 
	NOTE: Timely return to the care of the dam reduces early postoperative mortality.</li>
	<li>Monitor the animals for anesthesia by loss of the righting reflex hourly post-surgery for 3 h.</li>
	<li>Monitor the animals daily for 7 days for normal activity, food intake, and weight gain. Monitor the incision site for wound healing, closure, and reappearance of fur at the site of surgery. 
	NOTE: In the rare case that neurological changes such as seizures, central depression, or decreased appetite are observed during monitoring, euthanize the animal using intravascular perfusion or cervical dislocation under anesthesia.</li>
	<li>To prevent infection once the suture is closed and the wound is healing, apply topical triple-antibiotic at the incision site.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.3 mL insulin syringe</td>
			<td>BD Microfine + Insulin Syringe</td>
			<td>230-4533</td>
			<td>0.3-0.5 mL synringes will work</td>
		</tr>
		<tr>
			<td>1.5 mL microtube</td>
			<td>USA Scientific</td>
			<td>1615-5500</td>
			<td>Lot No. K194642H -3 511</td>
		</tr>
		<tr>
			<td>6-0 monofilament suture</td>
			<td>ETHICON</td>
			<td>667G</td>
			<td></td>
		</tr>
		<tr>
			<td>Analytical balance</td>
			<td>CCURIS Instruments</td>
			<td>W3200-320</td>
			<td></td>
		</tr>
		<tr>
			<td>Artificial CSF (aCSF)</td>
			<td>Tocris Bioscience</td>
			<td>3525</td>
			<td>Batch No: 72A</td>
		</tr>
		<tr>
			<td>Betadine</td>
			<td>Purdue Products L.P.</td>
			<td>301005-00</td>
			<td>NDC 67618-150-09</td>
		</tr>
		<tr>
			<td>Carprofen (injectable)</td>
			<td>Zoetis Inc.</td>
			<td>PI 4019448</td>
			<td>Rimadyl</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Decon Laboratories</td>
			<td>2701</td>
			<td></td>
		</tr>
		<tr>
			<td>Heating pad</td>
			<td>Sunbeam</td>
			<td>E12107-819</td>
			<td>UL 612A, Z-1228-001</td>
		</tr>
		<tr>
			<td>Hemoglobin</td>
			<td>MP Biomedicals</td>
			<td>100714</td>
			<td>LOT NO. SR02321</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Piramal Critical Care</td>
			<td>NDC 66794-017-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane vaporizer</td>
			<td>VETEQUIP</td>
			<td>911103</td>
			<td></td>
		</tr>
		<tr>
			<td>Light for stereotactic insturment</td>
			<td>Dolan-Jenner industries</td>
			<td>Fiber-Lite MI-150</td>
			<td></td>
		</tr>
		<tr>
			<td>Microinjection syringe pump</td>
			<td>World Precision Instruments</td>
			<td>MICRO21</td>
			<td>Serial 184034 T08K</td>
		</tr>
		<tr>
			<td>Oxygen</td>
			<td>Airgas Healthcare</td>
			<td>UN1072</td>
			<td>LOT NUMBER S1432080XA02</td>
		</tr>
		<tr>
			<td>Sprague Dawley rats</td>
			<td>Charles River Laboratories</td>
			<td>Strain code: 001</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereotactic instrument</td>
			<td>KOPF Instuments</td>
			<td>Model 900LS Lazy Susan</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile cotton tipped applicator</td>
			<td>Fischerbrand</td>
			<td>23-400-118</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical blade</td>
			<td>covetrus</td>
			<td>#10</td>
			<td></td>
		</tr>
		<tr>
			<td>Topical triple antibiotic</td>
			<td>Triple Antibiotic Ointment</td>
			<td>NDC 51672-2120-1</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Miller, B. A., et al. <a href="https://app.jove.com/v/63345">Modeling Neonatal Intraventricular Hemorrhage Through Intraventricular Injection of Hemoglobin.</a> J. Vis. Exp. (2022).
This video demonstrates a method to generate a rat model of neonatal intraventricular hemorrhage. In this procedure, an anesthetized rat pup is injected with hemoglobin into the lateral ventricle of the brain. The hemoglobin causes oxidative stress and releases heme, simulating intraventricular hemorrhage. The resulting damage to brain tissues, driven by reactive oxygen species and inflammatory cytokines, leads to ventricular enlargement, a common consequence of intraventricular hemorrhage.

Source: Miller, B. A., et al. Modeling Neonatal Intraventricular Hemorrhage Through Intraventricular Injection of Hemoglobin. J. Vis. Exp. (2022).
This ...

Secure an anesthetized rat pup on a stereotactic frame. Disinfect the head and incise to expose the skull.
Identify the bregma, an anatomical landmark, to locate the lateral ventricles, cerebrospinal fluid-filled cavities in the brain.
Choroid plexus epithelial cells and ependymal cells line the ventricles, forming a barrier from the periventricular space.
Use a microsyringe to inject hemoglobin into a ventricle, mimicking intraventricular hemorrhage.
Withdraw the needle, close the incision, and allow the pup to recover.
Injected hemoglobin undergoes degradation, releasing heme and generating reactive oxygen species or ROS.
ROS oxidize cell membrane lipids and proteins, causing cell death and disrupting the barrier, which allows heme to enter the periventricular space.
Tissue-resident immune cells recognize free heme as a danger signal and release proinflammatory cytokines to recruit additional immune cells, which damage the neighboring brain tissues.
The loss of periventricular tissue leads to ventricular enlargement, a consequence of intraventricular hemorrhage.

11 Views. Modeling Neonatal Intraventricular Hemorrhage Through Intraventricular Injection of Hemoglobin

Video: Modeling Neonatal Intraventricular Hemorrhage Through Intraventricular Injection of Hemoglobin - Experiment

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Modeling Neonatal Intraventricular Hemorrhage Through Intraventricular Injection of Hemoglobin

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Modeling Neonatal Intraventricular Hemorrhage Through Intraventricular Injection of Hemoglobin