The authors express their gratitude to the Department of Reproductive Medicine staff at Meizhou People&#39;s Hospital in Guangdong, China, for their active participation in this study.

All procedures were conducted in accordance with the standard operating procedures of the Department of Reproductive Medicine at Meizhou People&#39;s Hospital and were subject to review by the Ethics Committee of the same department. Written informed consent was obtained from each participating couple.The study included participants undergoing their first IVF cycle with cleavage or blastocyst stage embryo transfer, as well as females between 20 and 45 years of age. Exclusion criteria included the use of donor eggs/sperm and specific conditions such as congenital or secondary uterine abnormalities (e.g., septate uterus, unicornuate uterus, uterine didelphys), adenomyosis, uterine submucosal fibroids, or endometrial thickness below 7 mm on the day of embryo transfer. A total of 115 patients enrolled between December 2023 and February 2024 were stratified into two groups based on whether immediate partial removal of OCCC was performed.
1. Preparation before oocyte retrieval
<ol>
	<li>Place the oocyte pick-up dishes (Table of Materials) in the incubator at 37 &#176;C overnight.</li>
	<li>Incubate 12 mL of follicle manipulating medium (Table of Materials) in 14 mL round bottom test tubes at 37 &#176;C overnight. Transfer the tubes to the prewarmed test tube rack 30 min before oocyte retrieval.</li>
	<li>Prepare the follicle manipulating medium: Add 9 mL of follicle manipulating medium to a 14 mL round-bottom test tube (Table of Materials) and incubate at 37 &#176;C overnight. On the day of oocyte retrieval, transfer 4.5 mL of the medium to a preincubated oocyte pick-up dish (Table of Materials) covered with 2 mL of 100% paraffin oil.</li>
	<li>Prepare the OCCC flushing solution: Add 4.5 mL of fertilization medium (Table of Materials) to an oocyte pick-up dish and incubate at 37 &#176;C overnight.</li>
	<li>Prepare the fertilization medium: Add 1 mL of fertilization medium to a 6 cm dish covered with 100% paraffin oil (Table of Materials) and incubate at 37 &#176;C overnight.</li>
	<li>Prepare the glass Pasteur pipettes (Table of Materials): Gently blunt and round the tips by burning them on an alcohol lamp for about 5 s (Figure 1).Attach a suction head to the tip of the Pasteur pipettes, ensuring it can touch the bottom of the dish without scratching. Rinse the glass Pasteur pipettes and 10 cm dishes with a follicle manipulation medium.</li>
</ol>
2. Oocyte retrieval and immediate partial removal of OCCC
NOTE: Use a stereomicroscope during oocyte isolation and OCCC removal procedures. For optimal results, all procedures should be performed on a heating table with temperature control (37 &#176;C), and all equipment should be preheated 30 min before oocyte retrieval.
<ol>
	<li>Collect preovulatory follicular fluid during oocyte retrieval according to the ESHRE recommendations8.
	<ol>
		<li>Briefly, use a 17 G needle to retrieve the oocytes under transvaginal ultrasound guidance. The appropriate negative pressure ranges from 120-140 mmHg.</li>
		<li>Pour the follicular fluid collected in the 14 mL round-bottom test tube into a 10 cm dish (Table of Materials), maintaining a fluid depth of less than 0.5 cm. Place the dish on a constant temperature platform (37 &#176;C).</li>
	</ol>
	</li>
	<li>Observe the OCCC under a stereomicroscope to confirm the presence of oocytes and assess their maturity. 
	NOTE: The maturity of the OOOC is assessed based on the morphology of the cumulus cells and the visibility of certain oocyte structures9.</li>
	<li>Aspirate the OCCC using a blunt-ended Pasteur pipette. Tilt the 10 cm dish about 15 degrees and spread out all large OCCC. Avoid aspirating the oocyte and surrounding granulosa cells within a 2500 &#181;m radius. Cut excess granulosa cells with the Pasteur pipette along the longitudinal axis (Figure 2). The detailed steps are as follows:
	<ol>
		<li>Observe with the naked eye to see if there is a grayish translucent mucoid mass in the follicular fluid. If not found, quickly search for the grayish translucent mucoid mass by rotating clockwise from the outside to the inside under a stereomicroscope, the OCCC.</li>
		<li>Confirm whether there are oocytes in the OCCC and make a preliminary assessment of oocyte maturity.</li>
		<li>Then, pre-prepare a silicone aspiration pipette with a round flame-polished Pasteur pipette, aspirate the OCCC, and at the same time, gently raise the culture dish at the 9-10 o&#39;clock position with the left ring finger of the hand holding the 10 cm dish, tilting the culture dish slightly about 15&#176; to the left (placing the ring finger under the dish is approximately the required angle).</li>
		<li>While spitting out the aspirated OCCC at the 11 o&#39;clock position of the dish, gently elongate it horizontally to the right. Then, observe the size of the granulosa cells around the expanded OCCC. Subsequently, at the suitable position of the horizontally elongated OCCC, use the Pasteur pipette to make a quick and gentle descending cut on the excess granulosa cells from top to bottom.</li>
	</ol>
	</li>
	<li>Transfer the cut OCCC into a collection dish containing OCCC flushing solution. Pour the remaining follicular fluid into a sterile sample container (Table of Materials). Repeat these steps until all complexes are collected.</li>
</ol>
3. Semen preparation and in vitro insemination
<ol>
	<li>Collect the semen samples via ejaculation on the morning of oocyte retrieval. Evaluate sperm concentration, motility, and morphology under a light microscope following World Health Organization (WHO, 2010) criteria10. Then, transfer the sperm pellet to a new centrifuge tube and wash it twice in fertilization medium11. Incubate them at 6% CO2 and 37 &#176;C until needed.</li>
	<li>Inseminate OCCCs with 1 x 105 to 3 x 105 motile spermatozoa per milliliter in a single droplet approximately 2-3 h after retrieval in the fertilization dish (Table of Materials). Add 6-8 OCCC and 1 mL of the fertilization medium to the fertilization dish. 
	NOTE: After washing the sticky OCCC, they are transferred to the fertilization dish using a Pasteur pipette. Release one OCCC at a time, lifting the pipette gently off the liquid surface before releasing the next, ensuring that each OCCC is placed individually in the dish to prevent clustering.</li>
</ol>
4. Cumulus cells removal
<ol>
	<li>For short-term fertilization, follow the steps described below.
	<ol>
		<li>Co-incubate OCCCs with spermatozoa for4-6 h. After incubation, mechanically remove the cumulus cells surrounding the oocytes.</li>
		<li>Aspirate the oocytes using a pipette with an inner diameter slightly smaller than the oocytes to ensure complete removal of cumulus cells.</li>
		<li>Confirm fertilization by observing the presence of two polar bodies. Oocytes showing a second polar body are considered fertilized.</li>
		<li>Identify those cases that do not show the second polar body as fertilization failures, necessitating rescue ICSI.</li>
	</ol>
	</li>
	<li>For regular fertilization, after 18-20 h of co-incubation, remove cumulus cells using the pipettes for fertilization assessment.</li>
</ol>
5. Embryo culture
<ol>
	<li>Culture and monitor the fertilized oocytes for 3-5 days following oocyte retrieval. Assess fertilization at 20 h post-insemination to determine the number of pronuclei. Subsequently, culture each zygote individually in a medium droplet and transfer the embryos post-fertilization to a cleavage medium. 
	NOTE: An ideal embryo by the third day should consist of six or more equally-sized blastomeres and exhibit a fragmentation rate of less than or equal to 10%12.</li>
	<li>The decision to proceed with blastocyst culture depends on the patient&#39;s preferences and the specific circumstances, such as the quantity and quality of embryos obtained on day 3. Transfer the day 3 embryos to the blastocyst medium and assess on day 5. Use Gardner&#39;s scoring system to evaluate blastocysts, with high-quality blastocysts having scores of &#8805; 4 BB13.</li>
</ol>
6. Outcome measurements and statistical analysis
<ol>
	<li>Report continuous data as the mean &#177; standard deviation and express ordinal data as proportions. Perform statistical analysis using the Student&#39;s t-test or the chi-squared test in an appropriate data analysis software (here, IBM SPSS Statistics). Deem a significance level of P &#60; 0.05 as statistically significant.</li>
</ol>

Improving IVF Outcomes with Partial Granulosa Cell Removal Post-Retrieval

<ol>
	<li>Chen, C., Kattera, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rescue+ICSI+of+oocytes+that+failed+to+extrude+the+second+polar+body+6+h+post-insemination+in+conventional+IVF.">Rescue ICSI of oocytes that failed to extrude the second polar body 6 h post-insemination in conventional IVF.</a> Hum Reprod. 18 (10), 2118-2121 (2003).</li><li>Barlow, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fertilization+failure+in+IVF:+why+and+what+next.">Fertilization failure in IVF: why and what next.</a> Hum Reprod. 5 (4), 451-456 (1990).</li><li>Kuczy&#324;ski, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rescue+ICSI+of+unfertilized+oocytes+after+IVF.">Rescue ICSI of unfertilized oocytes after IVF.</a> Hum Reprod. 17 (9), 2423-2427 (2002).</li><li>He, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+early+cumulus+cells+removal+and+early+rescue+ICSI+on+pregnancy+outcomes+in+high-risk+patients+of+fertilization+failure.">Effect of early cumulus cells removal and early rescue ICSI on pregnancy outcomes in high-risk patients of fertilization failure.</a> Gynecol Endocrinol. 34 (8), 689-693 (2018).</li><li>Turathum, B., Gao, E. M., Chian, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+function+of+cumulus+cells+in+oocyte+growth+and+maturation+and+in+subsequent+ovulation+and+fertilization.">The function of cumulus cells in oocyte growth and maturation and in subsequent ovulation and fertilization.</a> Cells. 10 (9), 2292 (2021).</li><li>B&#225;rcena, P., Rodr&#237;guez, M., Obradors, A., Vernaeve, V., Vassena, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Should+we+worry+about+the+clock?+Relationship+between+time+to+ICSI+and+reproductive+outcomes+in+cycles+with+fresh+and+vitrified+oocytes.">Should we worry about the clock? Relationship between time to ICSI and reproductive outcomes in cycles with fresh and vitrified oocytes.</a> Hum Reprod. 31 (6), 1182-1191 (2016).</li><li>Telfer, E. E., Grosbois, J., Odey, Y. L., Rosario, R., Anderson, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Making+a+good+egg:+human+oocyte+health,+aging,+and+in+vitro+development.">Making a good egg: human oocyte health, aging, and in vitro development.</a> Physiol Rev. 103 (4), 2623-2677 (2023).</li><li>D'Angelo, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recommendations+for+good+practice+in+ultrasound:+oocyte+pick+up.">Recommendations for good practice in ultrasound: oocyte pick up.</a> Hum Reprod Open. 2019 (4), hoz025 (2019).</li><li>Hu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptomic+profiles+reveal+the+characteristics+of+oocytes+and+cumulus+cells+at+GV,+MI,+and+MII+in+follicles+before+ovulation.">Transcriptomic profiles reveal the characteristics of oocytes and cumulus cells at GV, MI, and MII in follicles before ovulation.</a> J Ovarian Res. 16 (1), 225 (2023).</li><li>Lu, W. H., Gu, Y. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insights+into+semen+analysis:+a+Chinese+perspective+on+the+fifth+edition+of+the+WHO+laboratory+manual+for+the+examination+and+processing+of+human+semen.">Insights into semen analysis: a Chinese perspective on the fifth edition of the WHO laboratory manual for the examination and processing of human semen.</a> Asian J Androl. 12 (4), 605-606 (2010).</li><li>Delos Santos, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Revised+guidelines+for+good+practice+in+IVF+laboratories+(2015).">Revised guidelines for good practice in IVF laboratories (2015).</a> Hum Reprod. 31 (2015), 685-686 (2016).</li><li>Nagy, Z. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pronuclear+morphology+evaluation+with+subsequent+evaluation+of+embryo+morphology+significantly+increases+implantation+rates.">Pronuclear morphology evaluation with subsequent evaluation of embryo morphology significantly increases implantation rates.</a> Fertil Steril. 80 (1), 67-74 (2003).</li><li>Gardner, D. K., Lane, M., Stevens, J., Schlenker, T., Schoolcraft, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reprint+of:+Blastocyst+score+affects+implantation+and+pregnancy+outcome:+towards+a+single+blastocyst+transfer.">Reprint of: Blastocyst score affects implantation and pregnancy outcome: towards a single blastocyst transfer.</a> Fertil Steril. 112 (4), e81-e84 (2019).</li><li>Esfandiari, N., Claessens, E. A., Burjaq, H., Gotlieb, L., Casper, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ongoing+twin+pregnancy+after+rescue+intracytoplasmic+sperm+injection+of+unfertilized+abnormal+oocytes.">Ongoing twin pregnancy after rescue intracytoplasmic sperm injection of unfertilized abnormal oocytes.</a> Fertil Steril. 90 (1), e5-e7 (2008).</li><li>Wetzels, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+co-culture+with+human+fibroblasts+on+human+embryo+development+in+vitro+and+implantation.">The effects of co-culture with human fibroblasts on human embryo development in vitro and implantation.</a> Hum Reprod. 13 (5), 1325-1330 (1998).</li><li>Kattera, S., Chen, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short+coincubation+of+gametes+in+in+vitro+fertilization+improves+implantation+and+pregnancy+rates:+a+prospective,+randomized,+controlled+study.">Short coincubation of gametes in in vitro fertilization improves implantation and pregnancy rates: a prospective, randomized, controlled study.</a> Fertil Steril. 80 (4), 1017-1021 (2003).</li></ol>

The authors declare no competing interests.

The immediate partial removal of granulosa cells can accelerate the early disintegration process of short-term fertilization, reduce the observation time for the second polar body in both short-term fertilization and conventional IVF and do not compromise subsequent embryo development. Currently, laboratories globally employ various strategies to mitigate fertilization disorders and low success rates. The preferred method involves removing granulosa cells for observing the second polar body 4-6 h post short-term fertiliz...

In the field of in vitro fertilization-embryo transfer (IVF-ET), the typical fertilization rate falls within the range of 60% to 80%1. However, approximately 4% to 16% of cases encounter either complete fertilization failure or low fertilization rates, presenting challenges in prediction2,3. To improve clinical pregnancy outcomes and decrease the occurrences of complete non-fertilization and low fertilization, the utilization of short-term fertilization in IVF procedures is on the rise4. This approach entails the prompt removal of granulosa cells to monitor the extrusion of the second polar body, assess fertilization status, and potentially conduct remedial intracytoplasmic sperm injection (ICSI). The bond between the oocyte cumulus cell complex (OCCC) and the oocyte is stronger during short-term fertilization compared to traditional overnight fertilization, thereby increasing the challenge of OCCC removal5. Factors such as an excess of oocyte numbers, large OCCC complexes, or inter-oocyte adhesions can lead to prolonged granulosa cell removal times during short-term fertilization, thereby extending the period that eggs remain outside the incubator4. To optimize the fertilization observation period, a modified approach post-egg retrieval has been devised, involving partial excision of granulosa cells from oocytes with significant OCCC complexes. This technique aids in retaining the granulosa cells surrounding the oocytes, preserving the integrity of the early oocyte structure6, and allowing these cells to continue supplying vital factors for oocyte maturation and subsequent fertilization-embryo binding7. 
Consequently, this study focuses on patients undergoing IVF-ET treatment at a reproductive medicine center as research participants, aiming to investigate the impact of an immediate partial granulosa cell mechanical excision method on the duration of procedures for both short-term fertilization and overnight fertilization monitoring while also evaluating outcomes of normal fertilization and late-stage development. This research intends to offer valuable insights for enhancing embryo laboratory procedures in IVF.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>G-IVFT PLUS</td>
			<td>Vitrolife</td>
			<td>10136</td>
			<td></td>
		</tr>
		<tr>
			<td>OVOIL</td>
			<td>Vitrolife</td>
			<td>10029</td>
			<td></td>
		</tr>
		<tr>
			<td>G-MOPS PLUS</td>
			<td>Vitrolife</td>
			<td>10130</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 3003 dish</td>
			<td>Corning</td>
			<td>353003</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 3001 dish</td>
			<td>Corning</td>
			<td>353001</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 2001 tube</td>
			<td>Corning</td>
			<td>352001</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 4013 cup</td>
			<td>Corning</td>
			<td>354013</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 3037 dish</td>
			<td>Corning</td>
			<td>351058</td>
			<td></td>
		</tr>
		<tr>
			<td>Pasteur pipette</td>
			<td>Darwin</td>
			<td>D1150-5P</td>
			<td></td>
		</tr>
	</tbody>
</table>

the immediate partial removal of cumulus-oocyte complexes: a refined approach for rapid observation of in vitro fertilization

Despite the rapid advancements in clinical and laboratory technologies for in vitro fertilization (IVF), a significant proportion (10%-15%) of patients continue to experience fertilization disorders, leading to low fertilization rates and the production of nonviable embryos. Short-term fertilization involves the early removal of granulosa cells to observe the extrusion of the second polar body, enabling the assessment of fertilization and early remedial measures to address low fertilization rates and complete fertilization failure. However, the observation of short-term fertilization in IVF is impeded by challenges such as excessively large unprocessed clusters of cumulus-oocyte complexes and adhesion between eggs, necessitating complex external procedures for granulosa cell removal, and prolonged observation duration. To tackle this issue, this study proposes a method of immediate partial removal of granulosa cells post egg retrieval. This approach streamlines subsequent stages of short-term fertilization and traditional fertilization monitoring, reduces the time needed for oocyte extrusion, minimizes the likelihood of external environmental impacts on the oocytes, and decreases the risk of missing the critical fertilization observation window. Consequently, the study yields novel clinical evidence aimed at enhancing the operational efficiency of embryonic laboratories in IVF.

In the group undergoing short co-incubation procedures, patients who received rescue were excluded. Out of 47 patients receiving short-term insemination, no significant differences were observed in terms of patients&#39; age and the number of retrieved oocytes (Table 1). The immediate partial removal of OCCC resulted in a loosening of the remaining OCCC around the oocytes (Figure 3), facilitating the detection of a second polar body. The average procedure time was significan...

Author Spotlight: Evaluating the Impact of Immediate Partial Removal of Cumulus-Oocyte Complexes on Fertilization Efficiency and Embryo Quality

Here, we present an optimized method for promptly removing a portion of cumulus-oocyte complexes following egg retrieval to expedite the IVF observation process, reducing the time required for granulosa cell removal, minimizing oocyte exposure to external elements, and does not hinder embryo development, ultimately improving the efficiency of IVF laboratory procedures.

The Immediate Partial Removal of Cumulus-Oocyte Complexes: A Refined Approach for Rapid Observation of In Vitro Fertilization

Despite the rapid advancements in clinical and laboratory technologies for in vitro fertilization (IVF), a significant proportion (10%-15%) of patients ...

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Research

JoVE Journal

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279 Views.  Meizhou People's Hospital. Here, we present an optimized method for promptly removing a portion of cumulus-oocyte complexes following egg retrieval to expedite the IVF observation process, reducing the time required for granulosa cell removal, minimizing oocyte exposure to external elements, and does not hinder embryo development, ultimately improving the efficiency of IVF laboratory procedures.

Video: Author Spotlight: Evaluating the Impact of Immediate Partial Removal of Cumulus-Oocyte Complexes on Fertilization Efficiency and Embryo Quality

Clinical Testing and Spinal Cord Removal in a Mouse Model for Amyotrophic Lateral Sclerosis (ALS)

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder resulting in progressive degeneration of motoneurons. Peak of onset is around 60 years for the sporadic disease and around 50 years for the familial disease. Due to its progressive course, 50% of the patients die within 30 months of symptom onset. In order to evaluate novel treatment options for this disease, genetic mouse models of ALS have been generated based on human familial mutations in the SOD gene, such as the SOD1 (G93A) mutation. Most important aspects that have to be evaluated in the model are overall survival, clinical course and motor function. Here, we demonstrate the clinical evaluation, show the conduction of two behavioural motor tests and provide quantitative scoring systems for all parameters. Because an in depth analysis of the ALS mouse model usually requires an immunohistochemical examination of the spinal cord, we demonstrate its preparation in detail applying the dorsal laminectomy method. Exemplary histological findings are demonstrated. The comprehensive application of the depicted examination methods in studies on the mouse model of ALS will enable the researcher to reliably test future therapeutic options which can provide a basis for later human clinical trials.

Clinical Testing and Spinal Cord Removal in a Mouse Model for Amyotrophic Lateral Sclerosis ALS

A mouse model for amyotrophic lateral sclerosis (ALS) is examined clinically and behaviorally. As a prerequisite for an accompanying immunohistological analysis the preparation of the spinal cord is depicted in detail.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder resulting in progressive degeneration of motoneurons. Peak of onset is around 60 ...

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

A Neuroscientific Approach to the Examination of Concussions in Student-Athletes

Concussions are occurring at alarming rates in the United States and have become a serious public health concern. The CDC estimates that 1.6 to 3.8 million concussions occur in sports and recreational activities annually. Concussion as defined by the 2013 Concussion Consensus Statement &#8220;may be caused either by a direct blow to the head, face, neck or elsewhere on the body with an &#8216;impulsive&#8217; force transmitted to the head.&#8221; Concussions leave the individual with both short- and long-term effects. The short-term effects of sport related concussions may include changes in playing ability, confusion, memory disturbance, the loss of consciousness, slowing of reaction time, loss of coordination, headaches, dizziness, vomiting, changes in sleep patterns and mood changes. These symptoms typically resolve in a matter of days. However, while some individuals recover from a single concussion rather quickly, many experience lingering effects that can last for weeks or months. The factors related to concussion susceptibility and the subsequent recovery times are not well known or understood at this time. Several factors have been suggested and they include the individual&#8217;s concussion history, the severity of the initial injury, history of migraines, history of learning disabilities, history of psychiatric comorbidities, and possibly, genetic factors. Many studies have individually investigated certain factors both the short-term and long-term effects of concussions, recovery time course, susceptibility and recovery. What has not been clearly established is an effective multifaceted approach to concussion evaluation that would yield valuable information related to the etiology, functional changes, and recovery. The purpose of this manuscript is to show one such multifaceted approached which examines concussions using computerized neurocognitive testing, event related potentials, somatosensory perceptual responses, balance assessment, gait assessment and genetic testing.

There is great variability in an individual&#8217;s risk for concussion and their corresponding recovery. A multifaceted approach to concussion evaluation is warranted; including baseline testing of athletes before participation in sport and timely evaluation post injury. The goal of this protocol is to provide an appropriate multifaceted approach to examine concussions.

Concussions are occurring at alarming rates in the United States and have become a serious public health concern. The CDC estimates that 1.6 to 3.8 ...

The Mouse Round-window Approach for Ototoxic Agent Delivery: A Rapid and Reliable Technique for Inducing Cochlear Cell Degeneration

Investigators have utilized a wide array of animal models and investigative techniques to study the mammalian auditory system. Much of the basic research involving the cochlea and its associated neural pathways entails exposure of model cochleae to a variety of ototoxic agents. This allows investigators to study the effects of targeted damage to cochlear structures, and in some cases, the self-repair or regeneration of those structures. Various techniques exist for delivery of ototoxic agents to the cochlea. When selecting a particular technique, investigators must consider a number of factors, including the induction of inadvertent systemic toxicity, the amount of cochlear damage produced by the surgical procedure itself, the type of lesion desired, animal survivability, and reproducibility/reliability of results. Currently established techniques include parenteral injection, intra-peritoneal injection, trans-tympanic injection, endolymphatic sac injection, and cochleostomy with perilymphatic perfusion. Each of these methods has been successfully utilized and is well described in the literature; yet, each has various shortcomings. Here, we present a technique for topical application of ototoxic agents directly to the round window niche. This technique is non-invasive to inner ear structures, produces rapid onset of reliably targeted lesions, avoids systemic toxicity, and allows for an intra-animal control (the contra-lateral ear). Results stemming from this approach have helped deeper understanding of auditory pathophysiology, cochlear cell degeneration, and regenerative capacity in response to an acute injury. Future investigations may use this method to conduct interventional studies involving gene therapy and stem cell transplantation to combat hearing loss.

Various methods exist for introducing ototoxic agents to the cochleae of animal models. Presented is a surgical protocol for delivery of ototoxic agents to the round window niche. The procedure is reliable, creates targeted intra-cochlear lesions, and avoids mechanical damage to the microarchitecture. Examination of cochlear self-repair/regeneration is possible.

Investigators have utilized a wide array of animal models and investigative techniques to study the mammalian auditory system. Much of the basic research ...

An Improved Method for Rapid Intubation of the Trachea in Mice

Despite some anatomical and physiological differences, mouse models continue to be an essential tool for studying human lung disease. Bleomycin toxicity is a commonly used model to study both acute lung injury and fibrosis, and multiple methods have been developed for administering bleomycin (and other toxic agents) into the lungs. However, many of these approaches, such as transtracheal instillation, have inherent drawbacks, including the need for strong anesthetics and survival surgery. This paper reports a quick, reproducible method of intratracheal intubation that involves mild inhaled anesthesia, visualization of the trachea, and the use of a surrogate spirometer to confirm exposure. As a proof of concept, 8-12 week old C57BL/6 mice were administered either 2.0 U/kg of bleomycin or an equivalent volume of PBS, and both damage and fibrotic endpoints were measured post-exposure. This procedure allows researchers to treat a large cohort of mice in a relatively short period with little expense and minimal post-procedure care.

This article presents a rapid and simple method for administering bleomycin directly into the mouse trachea via intubation. Key advantages of this method are that it is highly reproducible, easy to master, and does not require specialized equipment or lengthy recovery times.

Despite some anatomical and physiological differences, mouse models continue to be an essential tool for studying human lung disease. Bleomycin toxicity ...

A Novel Approach for the Administration of Medications and Fluids in Emergency Scenarios and Settings

The available routes of administration commonly used for medications and fluids in the acute care setting are generally limited to oral, intravenous, or intraosseous routes, but in many patients, particularly in the emergency or critical care settings, these routes are often unavailable or time-consuming to access. A novel device is now available that offers an easy route for administration of medications or fluids via rectal mucosal absorption (also referred to as proctoclysis in the case of fluid administration and subsequent absorption). Although originally intended for the palliative care market, the utility of this device in the emergency setting has recently been described. Specifically, reports of patients being treated for dehydration, alcohol withdrawal, vomiting, fever, myocardial infarction, hyperthyroidism, and cardiac arrest have shown success with administration of a wide variety of medications or fluids (including water, aspirin, lorazepam, ondansetron, acetaminophen, methimazole, and buspirone). Device placement is straightforward, and based on the observation of expected effects from the medication administrations, absorption is rapid. The rapidity of absorption kinetics are further demonstrated in a recent report of the measurement of phenobarbital pharmacokinetics. We describe here the placement and use of this device, and demonstrate methods of pharmacokinetic measurements of medications administered by this method.

This study presents a novel device that offers an easy route to quickly provide medications and fluids in patients with limited or difficult IV access. This small-diameter device is placed in the distal third of the rectum, allowing for ongoing medication and fluids administration.

The available routes of administration commonly used for medications and fluids in the acute care setting are generally limited to oral, intravenous, or ...

Re-Arterialized Rat Partial Liver Transplantation with an in vivo Vessel-Oriented 70% Hepatectomy

Split liver transplantation and living liver donor liver transplantation were developed in the clinic to utilize liver organs in a more efficient manner. To better understand the mechanism behind these surgical procedures, a rat partial liver transplantation (PLTx) model was established for relevant surgical studies. Because of the complexity of the rat PLTx model, a protocol with detailed descriptions is required. An article published previously reported a protocol in which ex vivo hepatectomy was used to achieve 50% rat PLTx. In contrast to this protocol, we introduced a re-arterialized PLTx with an in vivo 70% hepatectomy. An updated vessel-oriented hepatectomy was incorporated into the rat PLTx to refine the microsurgical procedure. The portal veins and hepatic arteries of the left lateral lobe and the median lobe were individually dissected and ligated before removal of the liver parenchyma, thereby decreasing the probability of bleeding in the remnant liver stump. Furthermore, an end-to-side vessel anastomosis between the common hepatic artery and the enlarged proper hepatic artery was introduced to re-arterialize the hepatic artery. By using this end-to-side vessel anastomosis technique, the diameter of the anastomosis was enlarged, thereby decreasing the difficulty of hand suture and maintaining a high rate of anastomotic patency. Moreover, the cuff anastomosis of the infrahepatic vena cava was slightly modified. A section of circumferential liver parenchyma around the vena cava of a recipient was preserved during cuff anastomosis to maintain the three-dimensional shape of the vascular lumen. This section of liver parenchyma was removed after completing the anastomosis. With this modification, the step involving placement of stay sutures was omitted, thereby further shortening the cuff anastomosis time. By using this protocol of rat PLTx, a low liver enzyme level, an intact liver lobular architecture and a high survival rate were achieved after microsurgery.

Re-Arterialized Rat Partial Liver Transplantation with an in vivo Vessel-Oriented 70% Hepatectomy

Here, a protocol involving re-arterialized rat partial liver transplantation is presented. Specifically, 70% liver was resected in vivo by using an updated technique of vessel-oriented hepatectomy. The hepatic artery was reconstructed in an end-to-side manner. The cuff technique was modified to shorten the anastomosis time of the infrahepatic vena cava.

Split liver transplantation and living liver donor liver transplantation were developed in the clinic to utilize liver organs in a more efficient manner. ...

An In Vitro Approach to Photodynamic Therapy

Photodynamic therapy (PDT) is a medical procedure that involves incubation of an exogenously applied photosensitizer (PS) followed by visible light photoactivation to induce cell apoptosis. The Federal Drug Administration has approved PDT for the treatment of actinic keratosis, and clinical guidelines recommend PDT as a treatment for certain non-melanoma skin cancers and acne vulgaris. PDT is an advantageous therapeutic modality as it is low cost, non-invasive, and associated with minimal adverse events and scaring. In the first step of PDT, a PS is applied and allowed to accumulate intracellularly. Subsequent light irradiation induces reactive oxygen species formation, which may ultimately lead to cell apoptosis, membrane disruption, mitochondrial damage, immune modulation, keratinocyte proliferation, and collagen turnover. Herein, we present an in vitro method to study PDT in an adherent cell line. This treatment protocol is designed to simulate PDT and may be adjusted to studying the use of PDT with various cell lines, photosensitizers, incubation temperatures, or photoactivation wavelengths. Squamous cell carcinoma cells were incubated with 0, 0.5, 1.0, and 2 mM 5-aminolevulinic acid (5-ALA) for 30 min and photoactivated with 417 nm blue light for 1,000 s. The primary outcome measure was apoptosis and necrosis, as measured by annexin-V and 7-aminoactinomycin D flow cytometry. There was a dose-dependent increase in cell apoptosis following thirty-minute incubation of 5-ALA. To achieve high inter-test validity, it is important to maintain consistent incubation and light parameters when performing in vitro PDT experiments. PDT is a useful clinical procedure and in vitro research may allow for the development of novel PSs, optimization of protocols, and new indications for PDT.

An In Vitro Approach to Photodynamic Therapy

Photodynamic therapy (PDT) is a medical procedure that involves incubation of an exogenously applied photosensitizer (PS) followed by visible light photoactivation to induce apoptosis. We present an in vitro PDT protocol designed to simulate PDT that may be used to study differences in PS incubation and light treatment parameters.

Photodynamic therapy (PDT) is a medical procedure that involves incubation of an exogenously applied photosensitizer (PS) followed by visible light ...

Complete and Partial Aortic Occlusion for the Treatment of Hemorrhagic Shock in Swine

Hemorrhage remains the leading cause of preventable deaths in trauma. Endovascular management of non-compressible torso hemorrhage has been at the forefront of trauma care in recent years. Since complete aortic occlusion presents serious concerns, the concept of partial aortic occlusion has gained a growing attention. Here, we present a large animal model of hemorrhagic shock to investigate the effects of a novel partial aortic balloon occlusion catheter and compare it with a catheter that works on the principles of complete aortic occlusion. Swine are anesthetized and instrumented in order to conduct controlled fixed-volume hemorrhage, and hemodynamic and physiological parameters are monitored. Following hemorrhage, aortic balloon occlusion catheters are inserted and inflated in the supraceliac aorta for 60 min, during which the animals receive whole-blood resuscitation as 20% of the total blood volume (TBV). Following balloon deflation, the animals are monitored in a critical care setting for 4 h, during which they receive fluid resuscitation and vasopressors as needed. The partial aortic balloon occlusion demonstrated improved distal mean arterial pressures (MAPs) during the balloon inflation, decreased markers of ischemia, and decreased fluid resuscitation and vasopressor use. As swine physiology and homeostatic responses following hemorrhage have been well-documented and are like those in humans, a swine hemorrhagic shock model can be used to test various treatment strategies. In addition to treating hemorrhage, aortic balloon occlusion catheters have become popular for their role in cardiac arrest, cardiac and vascular surgery, and other high-risk elective surgical procedures.

Here, we present a protocol demonstrating a hemorrhagic shock model in swine that uses aortic occlusion as a bridge to definitive care in trauma. This model has application in testing a wide range of surgical and pharmacological therapeutic strategies.

Hemorrhage remains the leading cause of preventable deaths in trauma. Endovascular management of non-compressible torso hemorrhage has been at the ...

A Metadata Extraction Approach for Clinical Case Reports to Enable Advanced Understanding of Biomedical Concepts

Clinical case reports (CCRs) are a valuable means of sharing observations and insights in medicine. The form of these documents varies, and their content includes descriptions of numerous, novel disease presentations and treatments. Thus far, the text data within CCRs is largely unstructured, requiring significant human and computational effort to render these data useful for in-depth analysis. In this protocol, we describe methods for identifying metadata corresponding to specific biomedical concepts frequently observed within CCRs. We provide a metadata template as a guide for document annotation, recognizing that imposing structure on CCRs may be pursued by combinations of manual and automated effort. The approach presented here is appropriate for organization of concept-related text from a large literature corpus (e.g., thousands of CCRs) but may be easily adapted to facilitate more focused tasks or small sets of reports. The resulting structured text data includes sufficient semantic context to support a variety of subsequent text analysis workflows: meta-analyses to determine how to maximize CCR detail, epidemiological studies of rare diseases, and the development of models of medical language may all be made more realizable and manageable through the use of structured text data.

We present a protocol and associated metadata template for the extraction of text describing biomedical concepts in clinical case reports. The structured text values produced through this protocol can support deep analysis of thousands of clinical narratives.

Clinical case reports (CCRs) are a valuable means of sharing observations and insights in medicine. The form of these documents varies, and their content ...