Name: neurodevelopmental reflex testing in neonatal rat pups
Uploaded: 2017-04-24T14:00:00.000+00:00
Duration: 9 min 35 s
Description: Watch this Scientific Journal Video about Neurodevelopmental Reflex Testing in Neonatal Rat Pups at JoVE.com

The authors wish to thank our funding agencies, which include NeuroDevNet (a National Centres of Excellence), the ALVA Foundation, the Women's and Children's Health Research Institute, and the University of Alberta.

The Animal Care and Use Committee, Health Sciences at the University of Alberta approved all animal studies.
Note: While this protocol may be adapted to other species and strains, this protocol is written for Long-Evans rats. These rats have been shown to have superior motor performances and visual acuity compared to other rodent strains. 25,26 The protocol for timed pregnancies, dietary supplementation, maternal inflammation, and neurodevelopmental reflexes is as follows.
1. Experimental Animals
<ol>
	<li>When rats arrive to the animal housing facility, refrain from any interaction for a day or two to allow them to acclimate to their new environment. Following acclimatization, handle the rats every day for approximately five days, or when the rats are no longer showing signs of stress. Human-induced stress can alter test results and lead to pregnancy loss.</li>
	<li>Once the rats are comfortable, breeding begins. Place two females and one male rat in a double decker breeding cage (h= 38 cm, 1,800 cm2 floor area) overnight (3 PM - 8 AM). These cages prevent stress and overcrowding. In the morning, using a transfer pipette, flush the vagina of the female rats with approximately 0.25 mL of saline. Transfer the solution into a microcentrifuge tube.</li>
	<li>Place the two females and male back into their respective conventional cages.</li>
	<li>View the solution collected in section 1.2 under a light microscope (100X) to determine which stage of the estrous cycle the rat is experiencing and whether or not sperm are present. If there are sperm, record the date as embryonic day (E1) (Figure 1). If pregnant, give the female her own cage on E14. 
	NOTE: The cells in the vaginal smear are clear, therefore use a low light setting. Ensure that the condenser is adjusted to its lowest setting. Adjust the brightness control to dim the light. If a brightness dial is available, set it to 1.</li>
</ol>
2. Dietary Supplementation
NOTE: This protocol is intended to assess the therapeutic effects of dietary broccoli sprout consumption during the last week of pregnancy. Grow the sprouts according to Wu et al. 27.
<ol>
	<li>Soak the broccoli sprouts seeds for 2 - 3 h. Spread the seeds out on a countertop seed sprouter box and set on a warm surface for germination.</li>
	<li>Water the seeds twice daily, once in the AM (8 - 9 AM) and once in the PM (3 - 4 PM).</li>
	<li>On day five, place the sprouts by a window for the day (sprouts should have two leaves each and will become green over the day).</li>
	<li>At the end of the day, harvest the sprouts by gently pulling the broccoli sprouts out of the sifted box and lay on a flat surface to dry.</li>
	<li>Once the sprouts are completely dried, weigh out 200 mg and place in a sealed plastic bag.</li>
	<li>Beginning on E14, feed each pregnant rat 200 mg of dried broccoli sprouts daily until PD21 (the day pups are weaned).</li>
</ol>
3. Inflammation
<ol>
	<li>To induce inflammation and reproduce white matter injury in offspring, inject pregnant rats with lipopolysaccharide (LPS) every 12 h on E19 and E20. On the day of injection, bring the cage into the lab from the housing facility. Allow the pregnant rat to acclimate and become calm for at least 1 h.&#160;If a rat shows clinical signs of permanent systemic or kidney damage, it will be euthanized immediately and removed from the study.&#160;</li>
	<li>Weigh the pregnant dam.</li>
	<li>Dissolve 200 &#181;g/kg of LPS (serotype 0127:B8) in 100 &#181;L of saline. Aspirate the solution into a 1 mL syringe equipped with a 30 G &#189; needle.
	<ol>
		<li>Use a new needle for each injection. Allow the solution to warm up for a few min to minimize discomfort.</li>
	</ol>
	</li>
	<li>Record the time and inject the pregnant dam intraperitoneally. Alternate injections between sides to ensure equal distribution. 
	NOTE: Machholz et al. describes proper restraint and injection procedures. 28</li>
</ol>
4. Developmental Reflex Tests
<ol>
	<li>On PD1 (day of birth), remove pups from the dam and record birth weights. Place the pups back into their respective cages and move them to the animal facility until PD3. This ensures maternal bonding between dam and pup.
	<ol>
		<li>Record the reflex testing for reliable assessment. Adjust the video camera so that it is at maximum shutter speed for frame-by-frame analyses (1/1,000 s). Position the video camera so that it is directly above or beside the rat pup and all materials used for each reflex. For example, for hindlimb placing, place the camera beside the rat pup to ensure the lifting and placing of the hindlimb is captured.</li>
		<li>For accelerated righting, place the camera above the rat pup and landing pad in order to record the ability of the pup to right in midair.</li>
	</ol>
	</li>
	<li>Record weights daily until the end of the experiment. On PD3, begin neurodevelopmental reflex testing (Figure 4). Move cages into a quiet room for a minimum of 1 h prior to testing to allow for acclimatization to the environment.</li>
	<li>Perform neurodevelopmental reflex testing at a consistent time each day as rats are nocturnal. For example, test between 9 AM - 12 PM daily. Keep the pups directly under a heating lamp or on a heat pad at all times, to maintain a stable body temperature of 36.5 &#176;C; rat pups of this age lose heat easily. Measure with a rectal temperature probe.&#160;If done efficiently, pups body temperatures do not change because they are not removed from the dam and litter long enough. NOTE: Monitor the temperature of heat lamp to ensure it does not exceed 37&#160;&#176;C.
	<ol>
		<li>Record the score of each reflex each day until a positive response is observed. A positive reflex response occurs when the pup is able to perform a task on two consecutive days. The date of the first occurrence of a positive response is used for quantification. No further testing is required following the positive response. Hence the end date for each reflex is variable.</li>
	</ol>
	</li>
	<li>Forelimb Grasping
	<ol>
		<li>Beginning on PD3, conduct the forelimb grasp reflex test. Place a blunt rod against the palm of each forepaw and apply light pressure manually. The light pressure should slightly displace the forepaw to ensure that contact is made and the pups can feel the rod. Grasping will appear as flexion of all digits around the rod. Successful acquisition of this reflex occurs when both forepaws grasp the rod for two days in a row.</li>
		<li>Score the forelimb grasp: 
		0 for no grasping 
		1 for successful grasping by one forepaw (left or right forepaw can be specified here) 
		2 for successful grasping by both forepaws</li>
	</ol>
	</li>
	<li>Hindlimb Grasping
	<ol>
		<li>Beginning on PD3, conduct the hindlimb grasp reflex test. Place a blunt rod against the sole of each hindpaw and apply light pressure manually. The light pressure should slightly displace the hindpaw to ensure that contact is made and the pups can feel the rod. Successful grasping of the rod appears as flexion of the digits around the rod. Successful acquisition of this reflex occurs when both hindpaws grasp the rod for two days in a row.</li>
		<li>Score the hindlimb grasp: 
		0 for no grasping 
		1 for successful grasping by one hindpaw (left or right hindpaw can be specified here) 
		2 for successful grasping by both hindpaws</li>
	</ol>
	</li>
	<li>Righting
	<ol>
		<li>Beginning on PD3, begin the righting reflex test. Firmly hold the pup in a supine position, with all four paws upright. Let go of the pup and immediately start the timer. Righting is achieved when the pup is able to flip/roll over onto all four paws, and each paw is perpendicular to the body. Give each pup a maximum of 15 s to achieve this goal.</li>
		<li>Score the righting: 
		0 for lying on the back (or 15 s for the maximum allocated time) 
		1 for lying on the side (left or right side can be specified here) or the ability of the pup to right but in the wrong posture (or 15 s for the maximum allocated time) 
		2 for successful righting and appropriate posture</li>
	</ol>
	</li>
	<li>Hindlimb placing
	<ol>
		<li>Beginning on PD4, begin the hindlimb placing reflex test. Hold the pup vertically in the air, by the torso. Gently stroke the dorsum of the hindpaw with a blunt surface (such as the edge of a table). A correct placing reflex is when the rat pup withdraws the stimulated hindlimb, followed by placement of the hindlimb down on that surface.</li>
		<li>Score the hindlimb placing reflex: 
		0 for no successes 
		1 for placing of one hindpaw (left or right hindpaw can be specified here) 
		2 for successful hindlimb placing for both hindpaws</li>
	</ol>
	</li>
	<li>Cliff Avoidance
	<ol>
		<li>Beginning on PD4, begin the cliff avoidance testing. Place the rat pup at the edge of a flat surface, such that the forepaws and snout of the pup are over the edge. The correct outcome is a protective response, where the rat pup turns away from the edge of the cliff.&#160;The experimenter&#8217;s hand or a foam landing is placed beneath the cliff to catch the pup from the fall. &#160;&#160;</li>
		<li>Score the cliff avoidance: 
		0 for no movement or falling off the edge 
		1 for attempts to move away from the cliff but with hanging limbs 
		2 for successful movement away from the cliff</li>
	</ol>
	</li>
	<li>Gait
	<ol>
		<li>Begin the evaluation of gait on PD6. Place pups in the center of a 15 cm diameter circle and allow the pup 30 s to complete the task.14 A successful gait is performed when the rat pup is able to move both forepaws outside the circle in less than 30 s.</li>
		<li>Score the gait as the time in seconds it takes the rat pup to move outside the circle. Record the time it takes the pup to move both forepaws outside the circle. Record 30 s if the pup is unable to complete the task within the given time</li>
	</ol>
	</li>
	<li>Auditory Startle
	<ol>
		<li>Begin the analysis of auditory startle on PD10. Present a loud noise (bell) directly over the pup to assess whether or not a startle response is present. A positive startle response is observed when the pup displays a &#39;jerking&#39; movement, away from the sound.</li>
		<li>Score the auditory startle: 
		(or no) for no startle response 
		(or yes) for a positive startle response</li>
	</ol>
	</li>
	<li>Posture
	<ol>
		<li>Begin posture analysis on PD12. Place pups on a non-slippery open surface, and observe the posture the pup has when moving. An immature posture is reflected by dragging of the abdomen when moving, and perpendicular pointing of both forepaws and hindpaws relative to the body. A mature posture is acquired when the pup can lift the abdomen from the surface and both forepaws and hindpaws are pointed straight, or parallel to the body, when moving.</li>
		<li>Score the posture: 
		0 for no movement 
		1 for immature posture when moving 
		2 for mature posture while moving</li>
	</ol>
	</li>
	<li>Eye Opening
	<ol>
		<li>Begin eye-opening analysis on PD12. Record the day both eyelids open: 
		0 for no visible eyes 
		1 for one visible eye (left or right side can be specified here) 
		2 for two visible eyes</li>
	</ol>
	</li>
	<li>Accelerated Righting
	<ol>
		<li>Beginning on PD14, begin accelerated righting testing. Place the pup in a supine position 30.48 centimeters above a foam landing. Drop the pup and observe its ability to right itself. Righting refers to when the pup is able to turn over to the prone position and land on its paws.</li>
		<li>Score the accelerated righting: 
		0 for falling on its back 
		1 for falling on its side (left or right side can be specified here) 
		2 for landing on its paws</li>
	</ol>
	</li>
</ol>
Note: Different types of quantification of the reflexes are available depending on the question of the investigators. Quantification of the reflexes can be done as initial observation of a reflex, first day of appearance up until the disappearance of the reflex, time it takes to successfully perform the task, speed of performance, and/or improvement of performance over time. 13, 14, 29,30,31 For the current study, the first day of appearance was used as a score. When using multiparous models, pups from the same litter typically behave similarly due to their genetic makeup, in utero and postpartum environment, and nutritional availability.32 Therefore, a single pup cannot be accounted for as an n = 1 due to litter bias. Pups examined within a litter can be averaged so that each litter represents an n = 1. 32, 33 Alternatively, several pups within the same litter can be analyzed using the mixed effects model, which takes into account pups within the same litter. 32, 33

<ol>
	<li>Farber, J. M., Shapiro, B. K., Palmer, F. B., Capute, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+diagnostic+value+of+the+neurodevelopmental+examination.">The diagnostic value of the neurodevelopmental examination.</a> Clin Pediatr (Phila. 24 (7), 367-372 (1985).</li><li>Zafeiriou, D. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primitive+reflexes+and+postural+reactions+in+the+neurodevelopmental+examination).">Primitive reflexes and postural reactions in the neurodevelopmental examination).</a> Pediatr. Neurol. 31 (1), 1-8 (2004).</li><li>Clancy, B., Finlay, B. L., Darlington, R. B., Anand, K. J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extrapolating+brain+development+from+experimental+species+to+humans.">Extrapolating brain development from experimental species to humans.</a> Neurotoxicology. 28 (5), 931-937 (2007).</li><li>Dobbing, J., Sands, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+aspects+of+the+brain+growth+spurt.">Comparative aspects of the brain growth spurt.</a> Early Hum. Dev. 3 (1), 79-83 (1979).</li><li>Semple, B. D., Blomgren, K., Gimlin, K., Ferriero, D. M., Noble-Haeusslein, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+development+in+rodents+and+humans:+Identifying+benchmarks+of+maturation+and+vulnerability+to+injury+across+species.">Brain development in rodents and humans: Identifying benchmarks of maturation and vulnerability to injury across species.</a> Prog. Neurobiol. , (2013).</li><li>Dobbing, J., Sands, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+growth+and+development+of+human+brain.">Quantitative growth and development of human brain.</a> Arch. Dis. Child. 48 (10), 757-767 (1973).</li><li>Tucker, A. M., Aquilina, K., Chakkarapani, E., Hobbs, C. E., Thoresen, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+amplitude-integrated+electroencephalography+and+interburst+interval+in+the+rat.">Development of amplitude-integrated electroencephalography and interburst interval in the rat.</a> Pediatr. Res. 65 (1), 62-66 (2009).</li><li>Dean, J. 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=Strain-specific+differences+in+perinatal+rodent+oligodendrocyte+lineage+progression+and+its+correlation+with.">Strain-specific differences in perinatal rodent oligodendrocyte lineage progression and its correlation with.</a> Dev. Neurosci. 33 (3-4), 251-260 (2011).</li><li>Back, S. A., Riddle, A., McClure, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maturation-dependent+vulnerability+of+perinatal+white+matter+in+premature+birth.">Maturation-dependent vulnerability of perinatal white matter in premature birth.</a> Stroke. 38, 724-730 (2007).</li><li>Back, S. 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=Late+oligodendrocyte+progenitors+coincide+with+the+developmental+window+of+vulnerability+for+human+perinatal+white+matter+injury.">Late oligodendrocyte progenitors coincide with the developmental window of vulnerability for human perinatal white matter injury.</a> J. Neurosci. 21 (4), 1302-1312 (2001).</li><li>Downes, N., Mullins, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+of+myelin+in+the+brain+of+the+juvenile+rat.">The development of myelin in the brain of the juvenile rat.</a> Toxicol. Pathol. 42 (5), 913-922 (2014).</li><li>Jakovcevski, I., Filipovic, R., Mo, Z., Rakic, S., Zecevic, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oligodendrocyte+development+and+the+onset+of+myelination+in+the+human+fetal+brain.">Oligodendrocyte development and the onset of myelination in the human fetal brain.</a> Front. Neuroanat. 3 (5), (2009).</li><li>Fox, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reflex-ontogeny+and+behavioural+development+of+the+mouse.">Reflex-ontogeny and behavioural development of the mouse.</a> Anim. Behav. 13 (2), 234-241 (1965).</li><li>Lubics, 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=Neurological+reflexes+and+early+motor+behavior+in+rats+subjected+to+neonatal+hypoxic-ischemic+injury.">Neurological reflexes and early motor behavior in rats subjected to neonatal hypoxic-ischemic injury.</a> Behav. Brain Res. 157 (1), 157-165 (2005).</li><li>Futagi, Y., Toribe, Y., Suzuki, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+grasp+reflex+and+moro+reflex+in+infants:+hierarchy+of+primitive+reflex+responses.">The grasp reflex and moro reflex in infants: hierarchy of primitive reflex responses.</a> Int. J. Pediatr. , 191562 (2012).</li><li>Hashimoto, R., Tanaka, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+the+supplementary+motor+area+and+anterior+cingulate+gyrus+to+pathological+grasping+phenomena.">Contribution of the supplementary motor area and anterior cingulate gyrus to pathological grasping phenomena.</a> Eur. Neurol. 40 (3), 151-158 (1998).</li><li>Isaza Jaramillo, S. 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=Accuracy+of+the+Babinski+sign+in+the+identification+of+pyramidal+tract+dysfunction.">Accuracy of the Babinski sign in the identification of pyramidal tract dysfunction.</a> J. Neurol. Sci. 343 (1-2), 66-68 (2014).</li><li>Donatelle, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+of+the+corticospinal+tract+and+the+development+of+placing+reactions+in+the+postnatal+rat.">Growth of the corticospinal tract and the development of placing reactions in the postnatal rat.</a> J. Comp. Neurol. 175 (2), 207-231 (1977).</li><li>Palanza, P., Parmigiani, S., vom Saal, F. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+Prenatal+Exposure+to+Low+Doses+of+Diethylstilbestrol,+o,p'DDT+and+Methoxychlor+on+Postnatal+Growth+and+Neurobehavioral+Development+in+Male+and+Female+Mice.">Effects of Prenatal Exposure to Low Doses of Diethylstilbestrol, o,p'DDT and Methoxychlor on Postnatal Growth and Neurobehavioral Development in Male and Female Mice.</a> Horm. Behav. 40 (2), 252-265 (2001).</li><li>Pantaleoni, G. C., 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=Effects+of+maternal+exposure+to+polychlorobiphenyls+(PCBs)+on+F1+generation+behavior+in+the+rat.">Effects of maternal exposure to polychlorobiphenyls (PCBs) on F1 generation behavior in the rat.</a> Fundam. Appl. Toxicol. 11 (3), 440-449 (1988).</li><li>Yeomans, J. S., Frankland, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+acoustic+startle+reflex:+neurons+and+connections.">The acoustic startle reflex: neurons and connections.</a> Brain Res. Rev. 21 (3), 301-314 (1995).</li><li>Vinay, L., Brocard, F., Clarac, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+maturation+of+motoneurons+innervating+ankle+flexor+and+extensor+muscles+in+the+neonatal+rat.">Differential maturation of motoneurons innervating ankle flexor and extensor muscles in the neonatal rat.</a> Eur. J. Neurosci. 12 (12), 4562-4566 (2000).</li><li>Geisler, H. C., Westerga, J., Gramsbergen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+posture+in+the+rat.">Development of posture in the rat.</a> Acta Neurobiol. Exp.(Wars. 53 (4), 517-523 (1993).</li><li>Heinen, K., 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=Gabaa+receptor+maturation+in+relation+to+eye+opening+in+the+rat+visual+cortex).">Gabaa receptor maturation in relation to eye opening in the rat visual cortex).</a> Neuroscience. 124 (1), 161-171 (2004).</li><li>Whishaw, I. Q., Gorny, B., Foroud, A., Kleim, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-Evans+and+Sprague-Dawley+rats+have+similar+skilled+reaching+success+and+limb+representations+in+motor+cortex+but+different+movements:+some+cautionary+insights+into+the+selection+of+rat+strains+for+neurobiological+motor+research.">Long-Evans and Sprague-Dawley rats have similar skilled reaching success and limb representations in motor cortex but different movements: some cautionary insights into the selection of rat strains for neurobiological motor research.</a> Behav. Brain Res. 145 (1-2), 221-232 (2003).</li><li>Prusky, G. T., Harker, K. T., Douglas, R. M., Whishaw, I. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variation+in+visual+acuity+within+pigmented,+and+between+pigmented+and+albino+rat+strains.">Variation in visual acuity within pigmented, and between pigmented and albino rat strains.</a> Behav. Brain Res. 136 (2), 339-348 (2002).</li><li>Wu, L., 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=Dietary+approach+to+attenuate+oxidative+stress,+hypertension,+and+inflammation+in+the+cardiovascular+system.">Dietary approach to attenuate oxidative stress, hypertension, and inflammation in the cardiovascular system.</a> Proc. Natl. Acad. Sci. U.S.A. 101 (18), 7094-7099 (2004).</li><li>Machholz, E., Mulder, G., Ruiz, C., Corning, B. F., Pritchett-Corning, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manual+restraint+and+common+compound+administration+routes+in+mice+and.">Manual restraint and common compound administration routes in mice and.</a> J. Vis. Exp. (67), (2012).</li><li>Rousset, C. I., 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=Maternal+exposure+to+lipopolysaccharide+leads+to+transient+motor+dysfunction+in+neonatal+rats.">Maternal exposure to lipopolysaccharide leads to transient motor dysfunction in neonatal rats.</a> Dev. Neurosci. 35 (2-3), 172-181 (2013).</li><li>Nguyen, A. T., Bahry, A. M. A., Shen, K. Q., Armstrong, E. A., Yager, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Consumption+of+broccoli+sprouts+during+late+gestation+and+lactation+confers+protection+against+developmental+delay+induced+by+maternal+inflammation.">Consumption of broccoli sprouts during late gestation and lactation confers protection against developmental delay induced by maternal inflammation.</a> Behav. Brain Res. 307, 239-249 (2016).</li><li>Black, A. M., Armstrong, E. A., Scott, O., Juurlink, B. J., Yager, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Broccoli+sprout+supplementation+during+pregnancy+prevents+brain+injury+in+the+newborn+rat+following+placental+insufficiency.">Broccoli sprout supplementation during pregnancy prevents brain injury in the newborn rat following placental insufficiency.</a> Behav. Brain Res. 291, 289-298 (2015).</li><li>Wainwright, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Issues+of+design+and+analysis+relating+to+the+use+of+multiparous+species+in+developmental+nutritional+studies.">Issues of design and analysis relating to the use of multiparous species in developmental nutritional studies.</a> J. Nutr. 128 (3), 661-663 (1998).</li><li>Lazic, S. E., Essioux, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+basic+and+translational+science+by+accounting+for+litter-to-litter+variation+in+animal+models.">Improving basic and translational science by accounting for litter-to-litter variation in animal models.</a> BMC Neurosci. 14, 14-37 (2013).</li><li>Sergio, D. P., Sanchez, S., Ruben, V. R., Ana, B. R., Barriga, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+behaviour+and+in+the+circadian+rhythms+of+melatonin+and+corticosterone+in+rats+subjected+to+a+forced-swimming+test.">Changes in behaviour and in the circadian rhythms of melatonin and corticosterone in rats subjected to a forced-swimming test.</a> J Appl Biomed. 1 (47), (2005).</li><li>Castelhano-Carlos, M., Baumans, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impact+of+light,+noise,+cage+cleaning+and+in-house+transport+on+welfare+and+stress+of+laboratory+rats.">The impact of light, noise, cage cleaning and in-house transport on welfare and stress of laboratory rats.</a> Lab. Anim. 43 (4), 311-327 (2009).</li><li>Zimmerberg, B., Ballard, G. A., Riley, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+of+thermoregulation+after+prenatal+exposure+to+alcohol+in+rats.">The development of thermoregulation after prenatal exposure to alcohol in rats.</a> Psychopharmacology (Berl. 91 (4), 479-484 (1987).</li><li>Fan, L. 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=Hypoxia-ischemia+induced+neurological+dysfunction+and+brain+injury+in+the+neonatal+rat).">Hypoxia-ischemia induced neurological dysfunction and brain injury in the neonatal rat).</a> Behav. Brain Res. 165 (1), 80-90 (2005).</li><li>Smart, J. L., Dobbing, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vulnerability+of+developing+brain.+VI.+Relative+effects+of+foetal+and+early+postnatal+undernutrition+on+reflex+ontogeny+and+development+of+behaviour+in+the+rat.">Vulnerability of developing brain. VI. Relative effects of foetal and early postnatal undernutrition on reflex ontogeny and development of behaviour in the rat.</a> Brain Res. 33 (2), 303-314 (1971).</li><li>Fox, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuro-Behavioral+ontogeny:+A+synthesis+of+ethological+and+neurophysiological+concepts.">Neuro-Behavioral ontogeny: A synthesis of ethological and neurophysiological concepts.</a> Brain Res. 2 (1), 3-20 (1966).</li><li>Piper, M. C., Mazer, B., Silver, K. M., Ramsay, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resolution+of+neurological+symptoms+in+high-risk+infants+during+the+first+two+years+of+life.">Resolution of neurological symptoms in high-risk infants during the first two years of life.</a> Dev. Med. Child Neurol. 30 (1), 26-35 (1988).</li><li>Shiotsuki, H., 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=A+rotarod+test+for+evaluation+of+motor+skill+learning.">A rotarod test for evaluation of motor skill learning.</a> J. Neurosci. Methods. 189 (2), 180-185 (2010).</li><li>Vorhees, C. V., Williams, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morris+water+maze:+procedures+for+assessing+spatial+and+related+forms+of+learning+and+memory.">Morris water maze: procedures for assessing spatial and related forms of learning and memory.</a> Nat. Protoc. 1 (2), 848-858 (2006).</li><li>Walf, A. A., Frye, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+the+elevated+plus+maze+as+an+assay+of+anxiety-related+behavior+in+rodents.">The use of the elevated plus maze as an assay of anxiety-related behavior in rodents.</a> Nat. Protoc. 2 (2), 322-328 (2007).</li></ol>

The authors have nothing to disclose.

Neurodevelopmental reflex testing is a predictive measure of abnormal cortical development and maturation, which may be of significance under circumstances where overt neuropathology is not evident. During neurodevelopmental testing, it is critical to ensure that pups are examined at the same time daily. Rats are nocturnal and therefore, their circadian rhythm may alter performance if testing is performed at different times during the day.34 Testing should be completed in a quiet room as loud nois...

Neurodevelopmental reflexes, or developmental milestones, are one of the earliest assessments used on human newborns and infants. Neurological reflexes are involuntary and repetitive movements that demonstrate brain stem and spinal cord reflexes. Maturation of higher cortical networks characterized by evolving migration, myelination, and synaptogenesis promote voluntary control and cortical inhibition. Alterations in the normal progression of the central nervous system evolution can disrupt brain development, resulting in abnormal cortical wiring, functioning, and myelination, causing neurodevelopmental reflex delays or absences. Human infants at high risk for neurodevelopmental disability often display abnormal early reflexes. Abnormal reflexes can present as a delay in acquisition, absence, prolonged presence, or reappearance later in life, and are predictive of developmental disabilities.1,2 Therefore, it is important to mimic reflex delays in experimental models of neurodevelopmental disabilities.
Rodents are commonly employed as experimental models. Rat pups are altricial when born, and therefore too immature to undertake specific or complex motor, sensory and/or cognitive behavioral tasks. In this regard, their developmental immaturity relates to both their physical and organ development. Rats are born hairless with an inability to thermoregulate, are blind, and unable to walk. With reference to brain development, substantial cortical maturation occurs postnatally. Newborn rat pups (day of birth referred to as postnatal day 1; PD1) have been suggested to reach a brain maturation level that is similar to a preterm human brain of 23 - 28 weeks gestation, whereas PD7-10 pups are equivalent to near-term human brain. 3,4,5,6 This correlation is based on gross anatomical analyses, however, other measures of brain maturation such as myelination and amplitude integrated electroencephalograms have also been described. 5,7 For example, pre-oligodendrocytes are the predominant cells in the developing human fetal brain from 23 - 32 weeks in utero, and this maturational stage corresponds to a PD1-3 rodent. 5,8,9,10 Moreover, myelination begins in utero in humans whereas in rat pups it appears in the forebrain around PD7-10; the newborn rodent brain remains largely un-myelinated. 11,12 Tucker et al. found that the amplitude integrated electroencephalogram pattern of a P1 rat to be similar to a 23-week gestation human fetus, whereas a PD7 and PD10 pup is akin to a 30 - 32 week and term infant, respectively.7 For these reasons, newborn reflex testing in neonatal rat pups provides an opportunity for capturing the ontogeny and/or disruption of brain development.
The battery of reflexes described below are adapted from studies by W.M. Fox and A. Lubics13,14 W.M. Fox was one of the earliest investigators with respect to the ontogeny of reflexes in the mouse. 13 These reflexes include, but are not limited to, limb grasping and placing, cliff avoidance, righting, accelerated righting, gait, auditory startle, posture, and eye opening. Both forelimb and hindlimb grasp (referred to as palmar and plantar grasp in humans, respectively) are facilitated by spinal reflexes and corticospinal inhibition from non-primary motor areas. 15,16 Hindlimb placing (plantar reflex) reflects maturation of the corticospinal tract. 16,17,18 Cliff avoidance (protective responses), righting (labyrinth), and accelerated righting involve integration and communication between sensory input and motor output (such as those involved with the vibrissae and vestibular systems). 19,20,21 Gait reflects locomotion.14 Auditory startle assesses acoustic stimulation and synaptic connections of giant neurons in the nucleus reticularis pontis caudalis.21 Posture involves appropriate cortical-spinal/spinal-cortical projections, muscle strength, and neuromuscular innervation. 22,23 Maturation of the gamma-aminobutyric acid receptors may correlate with eye opening.24 It is important to keep in mind that the reflexes reflect a much more complicated network and provided here is a general correlation. Moreover, these reflexes provide a quick and easy method of assessing neurological development at very young ages where more complex behavioral testing is not feasible.
The objective of this paper is to provide a general guideline for neurodevelopmental reflex testing that can be easily incorporated into experimental neonatal rat studies. The methodology described was carried out in Long-Evans neonatal rat pups and quantification of the results was based on first day of appearance. The day that reflex testing is initiated and the equipment utilized may be modified to better suit a different experimental model (such as for different strains and species). By establishing the normal physiological progression of reflex maturation in a specific animal model, investigators can evaluate the effects of external stressors, endogenous manipulations, and/or therapeutic interventions on neurodevelopment in neonatal rat models. Overall, the use of reflexes as a determination of brain maturity is advantageous in predicting perinatal brain injury, and is reflective of later neurodevelopmental outcomes.

<table><tbody><tr><td>&lt;em&gt;Breeding&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>Transfer pipettes</td><td>Fisherbrand</td><td>12-711-9AM</td><td>Used for vaginal flushes.</td></tr><tr><td>Sterile Saline</td><td>Hospira</td><td>7983254</td><td>The solution used to collect cells during vaginal flushes.</td></tr><tr><td>400 &amp;micro;L Microcentrifuge tubes</td><td>Fisherbrand</td><td>05-408-120</td><td>Used to hold the saline solution.</td></tr><tr><td>Light microscope</td><td>Leica</td><td>Leica ATC 2000</td><td>For observation of the saline solution. Can be any light microscope used in the lab.</td></tr><tr><td>Slides</td><td>Fisherbrand</td><td>12-552-5</td><td>The saline solution is placed on the slide. Can be any slides used in the lab.</td></tr><tr><td>Coverslips</td><td>Fisherbrand</td><td>12-545-F</td><td>To coverslip the slides. Can use any coverslips used in the lab.</td></tr><tr><td>&lt;em&gt;Dietary&amp;nbsp; Supplementation&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>Broccoli Sprouts seeds</td><td>Mumm&#039;s Sprouting Seeds</td><td></td><td>Broccoli sprouts seeds are ordered and grown in the lab.</td></tr><tr><td>Countertop Seed Sprouter Box</td><td>Mumm&#039;s Sprouting Seeds</td><td></td><td>A box is used to germinate and grow the seeds prior to harvest.</td></tr><tr><td>250 mL beaker</td><td></td><td></td><td>The beaker is used to soak the seed. Any size beaker that would fit can be used.</td></tr><tr><td>&lt;em&gt;Maternal Inflammation&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>Lipoplysaccharide (LPS)</td><td>Sigma</td><td>L3129</td><td>The endotoxin used to mimic maternal inflammation.</td></tr><tr><td>1 mL Syringe</td><td>BD Syringe</td><td>309659</td><td>Used to inject the pregnant rat.</td></tr><tr><td>Gauge (30 G x 1/2)</td><td>BD PrecisionGlide Needle</td><td>305106</td><td>Use the smallest needle to avoid pain and discomfort.</td></tr><tr><td>Sterile Saline (0.9% Sodium Chloride, USP)</td><td>Hospira</td><td></td><td>Saline is used to dissolve LPS.</td></tr><tr><td>&lt;em&gt;Weights&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>Scale</td><td>Denver Instrument</td><td></td><td>For recording the weights. Can be any scale with 2 decimal places used in the lab.</td></tr><tr><td>&lt;em&gt;Neurodevelopmental Reflexes&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>Thin blunt rod</td><td></td><td></td><td>Can be a paperclip or toothpick. This is for forelimb and hindlimb grasping.</td></tr><tr><td>Round filter paper</td><td>Whatman</td><td>1001 150</td><td>15 cm diameter paper used for gait analysis.</td></tr><tr><td>Timer</td><td>Fisher Scientific</td><td>06-662-51</td><td>For timing the time allocated to righting and gait.</td></tr><tr><td>Blunt surface</td><td></td><td></td><td>Can be an edge of a table. This is for hindlimb placing and cliff avoidance.</td></tr><tr><td>Foam landing</td><td></td><td></td><td>For when the pups perform accelerated righting.</td></tr><tr><td>Video recorder</td><td>Sony</td><td>VCT-D580RM</td><td>To record all reflexes tested. Must be able to record at 1/1,000 fps</td></tr><tr><td>Bell</td><td></td><td></td><td>For auditory startle.&amp;nbsp;</td></tr><tr><td>Heat lamp or pad</td><td></td><td></td><td>To maintain the body temperature of the pups underoing examination.</td></tr></tbody></table>

neurodevelopmental reflex testing in neonatal rat pups

Neurodevelopmental reflex testing is commonly used in clinical practice to assess the maturation of the nervous system. Neurodevelopmental reflexes are also referred to as primitive reflexes. They are sensitive and consistent with later outcomes. Abnormal reflexes are described as an absence, persistence, reappearance, or latency of reflexes, which are predictive indices of infants that are at high risk for neurodevelopmental disorders. Animal models of neurodevelopmental disabilities, such as cerebral palsy, often display aberrant developmental reflexes, as would be observed in human infants. The techniques described assess a variety of neurodevelopmental reflexes in neonatal rats. Neurodevelopmental reflex testing offers the investigator a testing method that is not otherwise available in such young animals. The methodology presented here aims to assist investigators in examining developmental milestones in neonatal rats as a method of detecting early-onset brain injury and/or determining the effectiveness of therapeutic interventions. The methodology presented here aims to provide a general guideline for investigators.

The timeline of this experimental design is presented in Figure 2. 30 The methods and results have previously been published. 30 The objective of the study was to assess whether dietary supplementation with broccoli sprouts during gestation and the preweaning period protected the offspring from neurodevelopmental delay induced by in utero exposure to LPS. Timed pregnant rats were given intraper...

Watch this Scientific Journal Video about Neurodevelopmental Reflex Testing in Neonatal Rat Pups at JoVE.com

Neurodevelopmental Reflex Testing in Neonatal Rat Pups

Behavioral testing is the gold standard for determining outcomes following brain injury, and can identify the presence of developmental disabilities in infants and children. Neurodevelopmental reflexes are an early indicator of these abnormalities. A host of easily accomplished developmental reflex tests in the neonatal rodent were developed and described here.

Neurodevelopmental reflex testing is commonly used in clinical practice to assess the maturation of the nervous system. Neurodevelopmental reflexes are ...

neurodevelopmental-reflex-testing-in-neonatal-rat-pups

Universidad San Sebastian

Research

JoVE Journal

Behavior

19.7K Views.  University of Alberta. Behavioral testing is the gold standard for determining outcomes following brain injury, and can identify the presence of developmental disabilities in infants and children. Neurodevelopmental reflexes are an early indicator of these abnormalities. A host of easily accomplished developmental reflex tests in the neonatal rodent were developed and described here. 

Video: Neurodevelopmental Reflex Testing in Neonatal Rat Pups

Assessment and Evaluation of the High Risk Neonate: The NICU Network Neurobehavioral Scale

There has been a long-standing interest in the assessment of the neurobehavioral integrity of the newborn infant. The NICU Network Neurobehavioral Scale (NNNS) was developed as an assessment for the at-risk infant. These are infants who are at increased risk for poor developmental outcome because of insults during prenatal development, such as substance exposure or prematurity or factors such as poverty, poor nutrition or lack of prenatal care that can have adverse effects on the intrauterine environment and affect the developing fetus. The NNNS assesses the full range of infant neurobehavioral performance including neurological integrity, behavioral functioning, and signs of stress/abstinence. The NNNS is a noninvasive neonatal assessment tool with demonstrated validity as a predictor, not only of medical outcomes such as cerebral palsy diagnosis, neurological abnormalities, and diseases with risks to the brain, but also of developmental outcomes such as mental and motor functioning, behavior problems, school readiness, and IQ. The NNNS can identify infants at high risk for abnormal developmental outcome and is an important clinical tool that enables medical researchers and health practitioners to identify these infants and develop intervention programs to optimize the development of these infants as early as possible. The video shows the NNNS procedures, shows examples of normal and abnormal performance and the various clinical populations in which the exam can be used.

The NICU Network Neurobehavioral Scale (NNNS) was developed as an assessment for the at-risk infant. The purpose of this article is to describe the NNNS, provide video examples of the NNNS procedures and discuss the ways in which the exam has been used.

There has been a long-standing interest in the assessment of the neurobehavioral integrity of the newborn infant.  The NICU Network Neurobehavioral Scale ...

Detecting Behavioral Deficits in Rats After Traumatic Brain Injury

With the increasing incidence of traumatic brain injury (TBI) in both civilian and military populations, TBI is now considered a chronic disease; however, few studies have investigated the long-term effects of injury in rodent models of TBI. Shown here are behavioral measures that are well-established in TBI research for times early after injury, such as two weeks, until two months. Some of these methods have previously been used at later times after injury, up to one year, but by very few laboratories. The methods demonstrated here are a short neurological assessment to test reflexes, a Beam-Balance to test balance, a Beam-Walk to test balance and motor coordination, and a working memory version of the Morris water maze that can be sensitive to deficits in reference memory. Male rats were handled and pre-trained to neurological, balance, and motor coordination tests prior to receiving parasagittal fluid percussion injury (FPI) or sham injury. Rats can be tested on the short neurological assessment (neuroscore), the beam-balance, and the Beam-Walk multiple times, while testing on the water maze can only be done once. This difference is because rats can remember the task, thus confounding the results if repeated testing is attempted in the same animal. When testing from one to three days after injury, significant differences are detected in all three non-cognitive tasks. However, differences in the Beam-Walk task were not detectable at later time points (after 3 months). Deficits were detected at 3 months in the Beam-Balance and at 6 months in the neuroscore. Deficits in working memory were detected out to 12 months after injury, and a deficit in a reference memory first appeared at 12 months. Thus, standard behavioral tests can be useful measures of persistent behavioral deficits after FPI.

The goal of the behavioral tests presented here is to detect functional deficits in rats after traumatic brain injury. Four specific tests are presented that detect deficits in behaviors to reflect the damage to specific brain areas at times extending to one year after injury.

With the increasing incidence of traumatic brain injury (TBI) in both civilian and military populations, TBI is now considered a chronic disease; however, ...

A Battery of Motor Tests in a Neonatal Mouse Model of Cerebral Palsy

As the sheer number of transgenic mice strains grow and rodent models of pediatric disease increase, there is an expanding need for a comprehensive, standardized battery of neonatal mouse motor tests. These tests can validate injury or disease models, determine treatment efficacy and/or assess motor behaviors in new transgenic strains. This paper presents a series of neonatal motor tests to evaluate general motor function, including ambulation, hindlimb foot angle, surface righting, negative geotaxis, front- and hindlimb suspension, grasping reflex, four limb grip strength and cliff aversion. Mice between the ages of post-natal day 2 to 14 can be used. In addition, these tests can be used for a wide range of neurological and neuromuscular pathologies, including cerebral palsy, hypoxic-ischemic encephalopathy, traumatic brain injury, spinal cord injury, neurodegenerative diseases, and neuromuscular disorders. These tests can also be used to determine the effects of pharmacological agents, as well as other types of therapeutic interventions. In this paper, motor deficits were evaluated in a novel neonatal mouse model of cerebral palsy that combines hypoxia, ischemia and inflammation. Forty-eight hours after injury, five tests out of the nine showed significant motor deficits: ambulation, hindlimb angle, hindlimb suspension, four limb grip strength, and grasping reflex. These tests revealed weakness in the hindlimbs, as well as fine motor skills such as grasping, which are similar to the motor deficits seen in human cerebral palsy patients.

Presented is a concise battery of mouse neonatal motor tests. Using these tests, neonatal motor deficits can be demonstrated in a variety of neonatal motor disorders. By having a standardized set of tests, results from different studies can be compared, allowing for better and accurate reporting between groups.

As the sheer number of transgenic mice strains grow and rodent models of pediatric disease increase, there is an expanding need for a comprehensive, ...

Medicine

The Use of Trace Eyeblink Classical Conditioning to Assess Hippocampal Dysfunction in a Rat Model of Fetal Alcohol Spectrum Disorders

Neonatal rats were administered a relatively high concentration of ethyl alcohol (11.9% v/v) during postnatal days 4-9, a time when the fetal brain undergoes rapid organizational change and is similar to accelerated brain changes that occur during the third trimester in humans. This model of fetal alcohol spectrum disorders (FASDs) produces severe brain damage, mimicking the amount and pattern of binge-drinking that occurs in some pregnant alcoholic mothers. We describe the use of trace eyeblink classical conditioning (ECC), a higher-order variant of associative learning, to assess long-term hippocampal dysfunction that is typically seen in alcohol-exposed adult offspring. At 90 days of age, rodents were surgically prepared with recording and stimulating electrodes, which measured electromyographic (EMG) blink activity from the left eyelid muscle and delivered mild shock posterior to the left eye, respectively. After a 5 day recovery period, they underwent 6 sessions of trace ECC to determine associative learning differences between alcohol-exposed and control rats. Trace ECC is one of many possible ECC procedures that can be easily modified using the same equipment and software, so that different neural systems can be assessed. ECC procedures in general, can be used as diagnostic tools for detecting neural pathology in different brain systems and different conditions that insult the brain.

Trace eyeblink classical conditioning (ECC) was used to assess hippocampal-dependent associative learning in adult rats that were administered a high concentration (11.9% v/v) of alcohol during early neonatal brain development. In general, ECC procedures are sound diagnostic tools for detecting brain dysfunction across many psychological and biomedical settings.

Neonatal rats were administered a relatively high concentration of ethyl alcohol (11.9% v/v) during postnatal days 4-9, a time when the fetal brain ...

Neuroscience

Recording EEG in Freely Moving Neonatal Rats Using a Novel Method

EEG is a useful method to detect electrical activity in the brain. Moreover, it is a widely used diagnostic tool for various neurological conditions, such as epilepsy and neurodegenerative disorders. However, it is technically difficult to obtain EEG recordings in neonates as it requires specialized handling and great care. Here, we present a novel method to record EEG in neonatal rat pups (P8-P15). We designed a simple and reliable electrode using computer pin loci; it can be easily implanted into the skull of a rat pup to record high-quality EEG signals in the normal and epileptic brain. Pups were given an intraperitoneal (i.p.) injection of the neurotoxin kainic acid (KA) to induce epileptic seizures. The surgical implantation performed in this procedure is less expensive than other EEG procedures for neonates. This method allows one to record high-quality and stable EEG signals for more than 1 week. Furthermore, this procedure can also be applied to adult rats and mice to study epilepsy or other neurological disorders.

Here, we introduce a novel technique designed to record electroencephalography (EEG) in freely moving neonatal epileptic pups and describe its procedures, features, and applications. This method allows one to record EEG for more than 1 week.

EEG is a useful method to detect electrical activity in the brain. Moreover, it is a widely used diagnostic tool for various neurological conditions, such ...

Neurobehavioral Assessments in a Mouse Model of Neonatal Hypoxic-ischemic Brain Injury

We performed unilateral carotid artery occlusion on CD-1 mice to create a neonatal hypoxic-ischemic (HI) model and investigated the effects of neonatal HI brain injury by studying neurobehavioral functions in these mice compared to non-operated (i.e., normal) mice. During the study, Rice-Vannucci&#39;s method was used to induce neonatal HI brain damage in postnatal day 7-10 (P7-10) mice. The HI operation was performed on the pups by unilateral carotid artery ligation and exposure to hypoxia (8% O2 and 92% N2 for 90 min). One week after the operation, the damaged brains were evaluated with the naked eye through the semi-transparent skull and were categorized into subgroups based on the absence (&#34;no cortical injury&#34; group) or presence (&#34;cortical injury&#34; group) of cortical injury, such as a lesion in the right hemisphere. On week 6, the following neurobehavioral tests were performed to evaluate the cognitive and motor functions: passive avoidance task (PAT), ladder walking test, and grip strength test. These behavioral tests are helpful in determining the effects of neonatal HI brain injury and are used in other mouse models of neurodegenerative diseases. In this study, neonatal HI brain injury mice showed motor deficits that corresponded to right hemisphere damage. The behavioral test results are relevant to the deficits observed in human neonatal HI patients, such as cerebral palsy or neonatal stroke patients. In this study, a mouse model of neonatal HI brain injury was established and showed different degrees of motor deficits and cognitive impairment compared to non-operated mice. This work provides basic information on the HI mouse model. MRI images demonstrate the different phenotypes, separated according to the severity of brain damage by motor and cognitive tests.

We performed unilateral carotid artery occlusion on postnatal day 7-10 CD-1 mouse pups to create a neonatal hypoxic-ischemic (HI) model and investigated the effects of HI brain injury. We studied neurobehavioral functions in these mice compared to non-operated normal mice.

We performed unilateral carotid artery occlusion on CD-1 mice to create a neonatal hypoxic-ischemic (HI) model and investigated the effects of neonatal HI ...

Electrophysiological Measurement of Noxious-evoked Brain Activity in Neonates Using a Flat-tip Probe Coupled to Electroencephalography

Pain is an unpleasant sensory and emotional experience. In non-verbal patients, it is very difficult to measure pain, even with pain assessment tools. Those tools are subjective or determine secondary physiological indicators which also have certain limitations particularly when exploring the effectiveness of analgesia. As cortical processing is essential for pain perception, brain activity measures may provide a useful approach to assess pain in infants. Here we present a method to assess nociception with electrophysiological brain activity recordings optimized for the use in newborn infants. To produce highly standardized and reproducible noxious stimuli we applied mechanical stimulation with a flat-tip probe, e.g., PinPrick, which is not skin-breaking and does not cause behavioral distress. The noxious-evoked potential allows the objective measurement of nociception in non-verbal patients. This method can be used in newborn infants as early as 34 weeks of gestational age. Moreover, it could be applied in different situations such as measuring the efficacy of analgesic or anesthetic drugs.

Measuring pain in non-verbal patients is a challenge. In this study we combine EEG recording with stimulation using a flat-tip probe to detect noxious-evoked brain activity in an objective manner.

Pain is an unpleasant sensory and emotional experience. In non-verbal patients, it is very difficult to measure pain, even with pain assessment tools. ...

Objective Nociceptive Assessment in Ventilated ICU Patients: A Feasibility Study Using Pupillometry and the Nociceptive Flexion Reflex

The concept of objective nociceptive assessment and optimal pain management have gained increasing attention. Despite the known negative short- and long-term consequences of unresolved pain or excessive analgosedation, adequate nociceptive monitoring remains challenging in non-communicative, critically ill adults. In the intensive care unit (ICU), routine nociceptive evaluation is carried out by the attending nurse using the Behavior Pain Scale (BPS) in mechanically ventilated patients. This assessment is limited by medication use (e.g., neuromuscular blocking agents) and the inherent subjective character of nociceptive evaluation by third parties.
Here, we describe the use of two nociceptive reflex testing devices as tools for objective pain evaluation: the pupillary dilation reflex (PDR) and nociception flexion reflex (NFR). These measurement tools are non-invasive and well tolerated, providing clinicians and researchers with objective information regarding two different nociceptive processing pathways: (1)&#160;the pain-related autonomic reactivity&#160;and (2) the ascending component of the somatosensory system. The use of PDR and NFR measurements are currently limited to specialized pain clinics and research institutions because of impressions that these are technically demanding or time-consuming procedures, or even because of a lack of knowledge regarding their existence.
By focusing on the two abovementioned nociceptive reflex assessments, this study evaluated their feasibility as a physiological pain measurement method in daily practice. Pursuing novel technologies for evaluating the analgesia level in unconscious patients may further improve individual pharmacological treatment and patient related outcome measures. Therefore, future research must include large well-designed clinical trials in a real-life environment.

Pain assessment in anesthetized patients who cannot communicate with the outside world in any way remains challenging despite the development of innovative objective pain evaluation tools. In this project, the pupillary dilation reflex and the nociception flexion reflex are assessed in critically ill, mechanically ventilated adult patients.

The concept of objective nociceptive assessment and optimal pain management have gained increasing attention. Despite the known negative short- and ...

Developmental Enhancement in Cerebral Palsy Rats via Spinal Manipulations

Enhancing the Development and Growth of Infant Cerebral Palsy Rats Using Selective Spinal Manipulations

Cerebral palsy (CP) is a refractory pediatric disease with a high prevalence, high disability rate, and difficult treatment. A variety of treatments are currently used for CP. The treatment involves drug and non-drug therapy. Traditional Chinese medicine external therapy is a very distinctive treatment method in non-drug therapy. As one of the external therapies of traditional Chinese medicine, massage is used in treating cerebral palsy and has good efficacy, small side effects, and strong operability. As a part of TCM external therapy, selective spinal manipulation can effectively promote the growth and development of infant rats with cerebral palsy.The operation was mainly divided into four steps: first, the rubbing method was applied to the spine and both sides of the spine for 1 min. The pressing and kneading method was applied to the spine for 5 min, and the muscles on both sides of the spine for 5 min. Second, pressing and kneading the sensitive local acupoints in the spine for 2 min were performed. Thirdly, the affected limb was treated by twisting method for 1 min. Fourth, the rubbing method was applied to a midline from the forehead to the back of the brain for 1 min. This study aimed to use selective spinal manipulation to treat infant rats with cerebral palsy. The weight, Rotarod test, Foot-fault score, and growth hormone of infant rats with cerebral palsy were detected to understand the effect of selective spinal manipulation on the growth and development of infant rats with cerebral palsy. The results showed that it can promote weight gain, improve balance ability and motor function, promote growth and development of infant cerebral palsy rats, promote growth hormone secretion, and increase the temperature of sensitive parts of the back.

This study illustrates the effect of selective spinal manipulation on the growth and development of infant rats with cerebral palsy, emphasizing the specific procedure and standardized protocol. Body weight measurement, Rotarod test, Foot-fault score, other behavioral tests, and growth hormone detection were performed to evaluate the protocol.

Cerebral palsy (CP) is a refractory pediatric disease with a high prevalence, high disability rate, and difficult treatment. A variety of treatments are ...

Creating a Preterm Rat Model for Early Postnatal Pain Research

A Preterm Rat Model for Pain Studies

This research delves into the consequences of consistent pinprick stimulation on preterm offspring to ascertain its long-term implications for pain sensitivity. The primary objective of this protocol was to investigate the impact of neonatal pinprick stimuli on the pain threshold in the later stages of life using a preterm rat model. By establishing this model, we aim to advance the research on understanding and managing early postnatal pain associated with prematurity. The findings of this study indicate that while the baseline thresholds to mechanical stimuli remained unaffected, there was a notable increase in mechanical hypersensitivity following complete Freund's adjuvant (CFA) injection in adult rats. Interestingly, compared with male rats, female rats demonstrated heightened inflammatory hypersensitivity. Notably, maternal behavior, the weight of the litters, and the growth trajectory of the offspring remained unchanged by the stimulation. The manifestation of altered nociceptive responses in adulthood after neonatal painful stimuli could be indicative of changes in sensory processing and the functioning of glucocorticoid receptors. However, further research is needed to understand the underlying mechanisms involved and to develop interventions for the consequences of prematurity and neonatal pain in adults.

Here, we present a concise protocol for creating a preterm rat model, facilitating research on early postnatal pain management. The method involves performing a cesarean section three days before the expected birth, extracting preterm rat pups via hysterectomy, and integrating them with the biological offspring of a surrogate dam.

This research delves into the consequences of consistent pinprick stimulation on preterm offspring to ascertain its long-term implications for pain ...