Effect of early life hydrocortisone administration on the neurobiology of the Limbic-Hypothalamic-Pituitory-Adrenal Axis

Varsha Bhatt-Mehta

Delia M Vazquez

DRAFT: 3/29/05

Introduction:

There is evidence to suggest that physical and psychological stress in early life can produce profound alterations in growth and development (1-3). The endocrine system most closely linked to stress in mammals is the limbic-hypothalamic-pituitary–adrenal axis (LPHA).The LPHA responds to stress by secreting glucocorticoids from the adrenal cortex. Among the functions of glucocorticoids are the mobilization of substrates for energy source, increased release of catecholamines, increased cardiovascular tone and suppression of nonessential systems for immediate survival such as immunity, growth and reproduction. In order to maintain stress responsiveness of the LPHA, both rapid activation and inhibition of the stress response is necessary as failure of either of these processes could affect permanently the growth and development of various systems in the mammal including the central nervous system(CNS).(4) Exogenously administered corticosteroids such as dexamethasone can inhibit the stress response of the LPHA axis for prolonged time periods and cause adverse effects on the growth and development.(5,6)

Dexamethasone, sometimes in prolonged courses of 28 days or more, has been used in the extremely low birth weight infants with severe respiratory distress syndrome to reduce the postnatal morbidity of chronic lung disease.(7-10) The time of use of dexamethasone has corresponded to 24-40 week gestational age, a time when the CNS undergoes profound structural and functional transformations.

In recent years, the importance of relative adrenal insufficiency and effects of critical illness on the LPHA axis in patients has been recognized.(11,12) The importance of LPHA axis to counter the inflammatory processes secondary to respiratory distress and sepsis in the newborn infant and the concept of relative adrenal insufficiency in neonates has also emerged recently. Use of low-dose hydrocortisone in extremely low birth weight infants increased the likelihood of survival without chronic lung disease.(13) While there are no well-controlled clinical studies to prove the effectiveness of hydrocortisone treatment for such insufficiency or convincing evidence of the presence of adrenal insufficiency, in clinical practice hydrocortisone therapy appears beneficial especially in patients dependent on vasopressors or unresponsive to high doses of vasopressors.(14)

As with dexamethasone, concerns remain as to the long term effects of hydrocortisone on the developing CNS in preterm and term neonates. The most challenging aspect of evaluating the effects of corticosteroids on the developing brain is the availability of an appropriate animal model that represents the developing human fetus. In recent years, animal models have been developed to evaluate the neurological effects and possible mechanisms of perinatal glucocorticoids on the developing brain.(16-18)

The purpose of this study is to evaluate the effects of hydrocortisone on the developing LPHA axis in a neonatal rat model developed at this institution.

Specific Objectives:

1. Evaluate the effects of a tapering dose of hydrocortisone on the development and function of the neonatal rat brain by evaluating the following when the animal is pre-adolescent:

-anxiety behavior utilizing open field and

-light -dark preference testingtests

-measurement of adrenocortical response to novelty stress (by measuring corticosterone and ACTH levels)and

- measurement of brain weights as a measure global effect before and after hydrocortisone treatment

Methods:

This study will be performed in neonatal rats.

Adult Sprague-Dawley rats will be housed and treated according to Guide for the Care and Use of Laboratory animals and mated using the trioone to one mating system (2F1F:1M). Pregnant females will be housed separately starting day 18 and the day of the birth of the pups designated as postnatal day one (PD1). On PD2, each litter will be sexed and culled to 12pups(6M:6F).On PD3, 3 pups representing both sexes will be assigned toand separated intoone of 3 four treatment groups within each litter on PD3 as shown below: 1) Handled Controls, 2) Vehicle Controls, 3) Dexamethasone (DEX), 4) Hydrocortisone (HC).

4 Treatment groups[MCIT1]

[MCIT2]

Within each group one animal will be sacrifieced at PD8 (see Brain Weight below) and the other two will be assigned to one of 5 time points needed for the stress response curve. We anticipate needing 160 animals to complete the studies proposed (see Animal needs below).

4 Treatment groups

144 animals will be needed for the whole experiment. Two litters will provide animals needed for the stress response curve.

Drug Treatment:

All pups will be removed from their mothers between the hours of 1100 and 1300 and handled for 5 minutes or treated as follows. Animals in the dexamethasone group will receive intramuscular Dexamethasone in a tapering dose on PD5 and PD6 (0.5mg/kg on PD5 and 0.1mg/kg on PD6). Animals in the Hydrocortisone group will also receive tapering doses of intramuscular hydrocortisone on PD5 and PD6 (5.0 mg/kg on PD5 and 1.0mg/kg on PD6). Animals in the vehicle group will receive equivalent volume of intramuscular sterile saline as the Dex /HC animals and animals in the handled group will receive no injection but will be handled during the same time on PD5 and PD6.

The following data will be collected on the PN days indicated.

Somatic

Animal measurementsGrowth: Length and weight will be measured before handling or injectionstreatments on PD PD 4 and 5 for each group, at weaning on PD 21 and prior to stress testing on PD 32. Length will be measured from the nose to the base of the tail in each pup.

Neurological assessment: Procedures for the neurological assessment on PD 7,14 and 20 will be adapted from the research by Altman etal, Fox et al and Wahlsten et al.(15-17) Two investigators will perform the evaluation using a scale of 0-5 (Appendix I) with a score of 5 corresponding to mature response. Neurodevelopment assessment in the neonate includes posture, righting reflex, postural flexion and extension, vibrissa placing, forelimb and hindlimb placing, geotaxis, and bar hold. Physical maturity will be measured by observing eye opening, ear opening, ear folding, fur development and tooth eruption.

Behavioral assessment: Behavioral response will occur at set times on PD21. Rats will be placed in the center of a brightly illuminated Plexiglas box with floor divided into 16 equal squares and motor and behavioral measures recorded during a 2-minute test period by observing their reaction to the squares.

Light-Dark preference test: The light dark preference will be tested on PD 33 using a covered 30x60x30-cm Plexiglas shuttle-box with a stainless steel grid floor suspended above the corncob bedding. Boxes will be divided into two equal sized compartments with a 12-cm wide opening. The light compartment will be constructed of white Plexiglas and brightly illuminated. The dark compartment will be constructed of black Plexiglas and minimally illuminated. Each animal will be placed in the dark compartment and the time elapsed before the animal enters the light side will be recorded. Locomotor activity as well as time spent in each compartment will be monitored by photocells located on the wall of each box, with the number of photocell beams interrupted per unit time recorded with microprocessor. Total testing time will be 5 minutes.

AdrenocorticalPituitary and Adrenocorticalresponse Response to novelty Noveltytest: On PD33 blood samples were collected via the tail nick method at 15,30,60 and 120 minutes following the 5 min novel exposure to the preference box with a basal level drawn prior to the procedure. Blood will be collected collected via decapitation and placed in tubes containing EDTA EDTA. Tubes will be and spun at 2000 rpm for 7 min. S and serum will be separated and stored at -200C until assayed for hormonal levels.

Corticosterone levels will be measured using a previously described radioimmunoassay method. (18). Plasma samples will be diluted 1:100 in 50 mM sodium phosphate buffer containing 2.5% bovine serum albumin at pH 7.5. Samples will be heated to separate corticosterone from the binding protein. The corticosterone body crossreacts 25 with cortisol and <1% with other endogenous steroids. Tritiated corticosterone will be used radiolabeled tracer. The bound [3H] corticosterone will be separated and measured.

Brain weights: Will be obtained during necropsy on PD8, 23, and 35 33 and quick frozen in isopentane for future analysis.

Table 1: Hydrocortisone and Dexamethsone Evaluation Schedule

PD5 / PD6 / PD7 / PD8 / PD14 / PD20 / PD21 / P33
D 0.5mg/kg
HC 5mg/kg / D 0.1mg/kg
HC 1mg/kg
Neuro / Brain weights / Neuro / Neuro / Light-Dark preferences
Weight
Length / Weight
Length / Weight
Length / Brain weight
Open field behavior / Adrenal Stress response . ACTH and Corticosterone levels
From all?

Animals Needed: We base the number of animals needed on the hormonal analysis. Eight animals are needed per time point. Therefore, 160 animals will be needed to complete the experiments planned (5 time points (0, 15, 30, 60, 120 min) x 4 groups (HAND, VEH, DEX, HC) x 8 animals= 160).

Statistical analysis:

Body weight, length and rate of growth, brain weight, hormonal values and behavioral data will be analyzed using descriptive statistics. Multivariate analysis with ANOVA test will allow for evaluating the relationship between sex, age and treatment simultaneously. The somatic growth data will be subjected to a repeated measure ANOVA analysis. Total and individual neurological scores will be averaged across groups using nonparametric test (Kruskal-Wallis).

REFERENCES:

  1. Dachir S, Kadar, T, Robinson, B. Levy, A. Cognitive deficits induced in young rats by long-term corticosterone administration. Behavior and Neural Biology 1993;60, 103-109.
  2. Duncan DF. Life stress as a precursor to adolescent drug dependence. International Journal of Addiction 1977;12:1047-1056.
  3. Henry C, Kabbaj M, Simon H, Le Moal, M, Maccari S. Prenatal stress increases the hypothalamo-pituitary-adrenal axis response in young and adult rats. Journal of Neuroendocrinology 1994;6:341-345.
  4. De Kloet ER, Rosenfeld P, Van Eelelen AM, Sutanto W, Levine S. stress, glucocorticoids and development. Progress in Brain Research 1988;73:101-120.
  5. Flagel SB, Vazquez DM, Watson S, Jr., Neal CR, Jr. Effects of tapering neonatal dexamethasone doses on rat growth, neurodevelopment, and stress response. American Journal of Physiology and Regulatory Integrative Comprehensive Physiology 2002;282: R55-R63.
  6. Alkalay AL, Klein AH, Nagel RA, Pomerance JJ. Evaluation of hypothalamic-pituitary-adrenal axis in premature infants treated with dexamethasone. American Journal of Perinatology 1996;13:473-477.
  7. Avery GB, Fletcher M, Kaplan M, Brudno DS. Controlled trial of Dexamethsone in respiratory-dependent infants with bronchopulmonary dysplasia. Pediatrics 1985;75:106-111.
  8. KazziNJ, Brans YW, Poland RL. Dexamethasone effects on the hospital course of infants with bronchopulmonary dysplasia who are dependent on artificial ventilation. Pediatrics 1990;86:722-727.
  9. Cummings JJ, D’Eugenio DB, Gross SJ. A controlled trial of dexamethasone in preterm infants at high risk for bronchopulmonary dysplasia. New England Journal of Medicine 1989;320:1505-1510.
  10. Kothadia JM, O’Shea TM, Roberts D, Auringer ST, Weaver RG, Dillard RG. Randomized, placebo-controlled trial of a 42-day tapering course of dexamethasone to reduce the duration of ventilatory dependency in very low birth weight infants. Pediatrics 1999;104:22-27.
  11. Cooper MS, Stewart PM. Corticosteroid Insufficiency in acutely Ill Patients. New England Journal of Medicine2003;348:727-734.
  12. Annane D, Bellissant E, Bollaert PE, Briegel J, Keh D, Kupfer Y. Corticosteroids for severe sepsis and septic shock: a systematic review and meta-analysis. British Medical Journal 38181.482222.55, august 2004.
  13. Watterberg KL, Gerdes JS, Gifford KL, Lin H-M. Prophylaxis against early adrenal insufficiency to prevent chronic lung disease in premature infants. Pediatrics 1999;104:1258-1263
  14. Baker CF, Schumacher RE, Barks J, Vazquez DM, Bhatt-Mehta, V. Evaluation of Hydrocortisone in Neonates with Pressor Resistant Hypotension. 2005 Pediatric Academic Societies’ Annual Meeting (Society for Pediatric Research) , Washington, DC, May 14-17.
  1. Altman J, McCrady B. The influence of nutrition on neural and behavioral development. IV. Effects of infantile undernutrition on the growth of the cerebellum. Developmental Psychobiology 1972:2:111-122.
  2. Fox WM. Reflex-ontogeny and behavioral development of the mouse. Animal Behavior 1965;13:234-241.
  3. Wahlsten D. A developmental timescale for postnatal changes in brain and behavior of the B6D2F2 mice. Brain Research 1974;72:251-264.
  4. Vazquez DM, Morano MI, Lopez JF, Watson SF, Akil H. Short-term adrenolactomy increases glucocorticoid and mineralocorticoid mRNA in selective areas of the developing hippocampus. Molecular and Cellular Neuroscience. 1993;4:455-471.

APPENDIX I

Table 1. Neurological exam rating scale

Parameter# / Behavior / 0 / 1 / 2 / 3 / 4 / 5
1 / Posture / Full Flexion / Partial flexion / Full extension / Partial extension / Normal
2 / Eye opening / Closed / Occasional opening / Occasional opening / Fully open
3 / Ear opening / Closed / Occasional opening / Occasional opening / Fully open
4 / Ear folding / Barely visible / Completely folded / Partially folded / Fully open / Fully open
5 / Fur development / No fur / Fine hairs / Partial fur / Mostly fur / Full fur
6 / Maxilliary eruption / No teeth / Initial maxillary / Partial maxillary / Partial maxillary / Mostly full / Maxilliary full
7 / Mandibular eruption / No teeth / Initial mandibular / Partial mandibular / Partial mandibular / Mostly full / Mandibular full
8 / Ear twitch response / No response / Weak response / Moderateresponse / Strong response
9 / Righting reflex / On side full flexion / Partial flexion / Extension predominates / Extension predominates / Partial extension / Normal position
10 / Postural flexion / Absent / Partial / Moderate / Present
11 / Postural extension / Absent / Partial / Moderate / Present
12 / Vibrissa placing / No response / Weak response / Moderate response / Strong response
13 / Forelimb placing / No response / Weak response / Moderate response / Strong response
14 / Hindlimb placing / No response / Weak response / Moderate response / Strong response
15 / Geotaxis / No response / Pivoting predominates / Turns 1800 / Strong response
16 / Bar hold / No grasp / Weak grasp / Moderate grasp / Full grasp
17 / Grasp reflex forelimb / No grasp / Weak grasp / Moderate grasp / Full grasp
18 / Grasp reflex hindlimb / No grasp / Weak grasp / Moderate grasp / Full grasp
19 / Cross extensor / Absent / Weak / Moderate / Strong

To evaluate responses of rat pups to neurological test battery, a score of 0 represents complete immature response on exam or immature physical development. A score of 5 represents completely mature response or physical maturation.

Parameters 1-7 = Physical maturation

Parameters 8-19 = Neurodevelopmental assessment parameters

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[MCIT2]Consider the scheme below- with figure legend