Neurodevelopmental Reflex Testing in Neonatal Rat Pups

摘要

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.

摘要

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.

引言

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. Lubics^13,14 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.

方案

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

1. 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.
2. 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.
3. Place the two females and male back into their respective conventional cages.
4. 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.

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.

1. 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.
2. Water the seeds twice daily, once in the AM (8 - 9 AM) and once in the PM (3 - 4 PM).
3. 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).
4. 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.
5. Once the sprouts are completely dried, weigh out 200 mg and place in a sealed plastic bag.
6. Beginning on E14, feed each pregnant rat 200 mg of dried broccoli sprouts daily until PD21 (the day pups are weaned).

3. Inflammation

1. 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. If a rat shows clinical signs of permanent systemic or kidney damage, it will be euthanized immediately and removed from the study. 
2. Weigh the pregnant dam.
3. Dissolve 200 µg/kg of LPS (serotype 0127:B8) in 100 µL of saline. Aspirate the solution into a 1 mL syringe equipped with a 30 G ½ needle.
   1. Use a new needle for each injection. Allow the solution to warm up for a few min to minimize discomfort.
4. 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

4. Developmental Reflex Tests

1. 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.
   1. 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.
   2. 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.
2. 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.
3. 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 °C; rat pups of this age lose heat easily. Measure with a rectal temperature probe. 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 °C.
   1. 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.
4. Forelimb Grasping
   1. 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.
   2. 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
5. Hindlimb Grasping
   1. 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.
   2. 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
6. Righting
   1. 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.
   2. 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
7. Hindlimb placing
   1. 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.
   2. 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
8. Cliff Avoidance
   1. 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. The experimenter’s hand or a foam landing is placed beneath the cliff to catch the pup from the fall.   
   2. 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
9. Gait
   1. 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.
   2. 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
10. Auditory Startle
    1. 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 'jerking' movement, away from the sound.
    2. Score the auditory startle:
       (or no) for no startle response
       (or yes) for a positive startle response
11. Posture
    1. 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.
    2. Score the posture:
       0 for no movement
       1 for immature posture when moving
       2 for mature posture while moving
12. Eye Opening
    1. 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
13. Accelerated Righting
    1. 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.
    2. 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

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}

结果

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...

讨论

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...

材料

本文使用的材料清单

姓名	公司	目录编号	评论
Breeding
移液管	Fisherbrand	12-711-9AM	用于阴道冲洗。
无菌盐水	Hospira	7983254	在阴道冲洗期间收集细胞的溶液。
400 和微型;L 微量离心管	Fisherbrand	05-408-120	用于盛放盐水溶液。
光学显微镜		Leica Leica ATC 2000	用于观察盐溶液。可以是实验室中使用的任何光学显微镜。
载玻片	Fisherbrand	12-552-5	将盐水溶液置于载玻片上。可以是实验室中使用的任何载玻片。
盖玻片	Fisherbrand	12-545-F	盖玻片。可以使用实验室中使用的任何盖玻片。
饮食 补充剂
西兰花芽种子	Mumm's 发芽种子		西兰花芽种子是在实验室订购和种植的。
台面种子发芽盒	Mumm's Sprouting Seeds		一个盒子用于在收获前发芽和生长种子。
250 mL 烧杯			烧杯用于浸泡种子。可以使用任何适合的尺寸烧杯。
母体炎症
脂溶脂糖 （LPS）	Sigma	L3129	用于模拟母体炎症的内毒素。
1 mL 注射器	BD 注射器	309659	用于注射怀孕的大鼠。
规格 （30 G x 1/2）	BD PrecisionGlide 针	305106	使用最小的针头以避免疼痛和不适。
无菌盐水（0.9% 氯化钠，USP）	Hospira		盐水用于溶解 LPS。
Weights
Scale	Denver Instrument		用于记录重量。可以是实验室中使用的任何刻度，小数点后 2 位。
神经发育反射
细钝棒			可以是回形针或牙签。这是为了抓握前肢和后肢。
圆形滤纸	Whatman	1001 150	直径为 15 厘米的纸，用于步态分析。
计时器	Fisher Scientific	06-662-51	用于计时分配给扶正和步态的时间。
钝面			可以是桌子的边缘。这是为了放置后肢和避开悬崖。
泡沫着陆			当幼崽进行加速扶正时。
录像机	Sony	VCT-D580RM	记录所有反射测试。必须能够以 1/1,000 fps 的
Bell			录制 用于听觉惊吓。
加热灯或垫			保持接受检查的幼犬的体温。