09202013 CANDLE SLLS Hair Cortisol KJSA&CRR

CONDITIONS AFFECTING NEUROCOGNITIVE

DEVELOPMENT AND LEARNING IN EARLY

CHILDHOOD

Cynthia R. Rovnaghi, MS Research Specialist & Manager, Pain Neurobiology Laboratory

University of Tennessee Health Science Center, Memphis.

K. J. S. “Sunny” Anand, MBBS, D.Phil. Professor of Pediatrics, Anesthesiology, Anatomy & Neurobiology

University of Tennessee Health Science Center, Memphis.

http://www.candlestudy.org http://www.theurbanchildinstitute.org http://www.uthsc.edu/

With regard to this presentation, Ms. Rovnaghi and Dr. Anand

have no Financial Relationships to disclose or Conflicts of Interest to resolve.

Amsterdam, The Netherlands

September 23-25, 2013

http://www.theurbanchildinstitute.org




Fetal programming of the HPA axis

Adapted from Kyrou & Weickert. Chapter 13,

Figures 1, 4, 13. www.endotext.org

Maternal exposure to

stress regulates the

fetal HPA axis

Cortisol is expressed

by the fetus

Maternal and fetal

levels increase during

gestation

Circadian cortisol

rhythms are present at

birth or soon thereafter

Widespread genomic

regulation by Cortisol

Response Elements

(CREs)




http://www.endotext.org/


Cortisol Imposed Changes in Brain

Development? • Prenatal and postnatal connectivity exceeds that of

adults (Stiles & Jernigan 2010; Stiles 2008)

• Activity-dependent pruning of developing synapses (Gogtay et al 2004; Sowell et al 2004; Ostby et al 2009; Stoneham et al 2010)

• Enriched environments enhance synaptogenesis (Kolb & Gibb 1991; Moncek et al 2004; Champagne et al 2007)

• Increased cortisol exposure reduces limbic system

brain volume linked to later cognitive/behavioral

disorders (Lupien et al. 1998; Wiedenmayer et al. 2006; Jorge et al. 2007;

Schechter et al. 2012; Moser et al. 2013)

• Anxiety, attention deficit, internalizing and

externalizing behaviors by 3 years (Kryski et al 2013)





Stress: A Matter of Perspective? Pain – Nociception

Scales

Numeric,1–10 for rating

Faces

Stress vs. Allostasis




http://www.bing.com/images/search?q=Pain+scale&id=E68E4E4CC7C6DB1361D787139105260C703A31E1&FORM=IQFRBA


Overarching Hypothesis

• Hair Cortisol (biomarker for ongoing stress during early development) will be altered by:

• Exposure to prenatal stress

• Ongoing maternal stress

• Maternal characteristics

• Infant characteristics





CANDLE Study: Hair Cortisol

• Cross sectional analysis of hair samples (3-50 mg)

obtained at Clinic Visits: CV1 (N=374), CV2 (N=

308), CV3 (N=190)

• Hair taped at distal end, 1-3 cm sample is cut close

to the scalp

• Clean, free of oils, waxes, any other hair products

• Analyzed after multiple protein extractions

ELISA-based assay system (ALPCO Diagnostics, Inc.)

• Intra-assay coefficients of variation: <5%

• Inter-assay coefficients of variation: <8%





Potential predictors • Outcome – Ln-Cortisol ng/mg

Demographics Child characteristics Maternal Race (Black vs. White)

Child's gender (female vs. male)

Maternal Education (< High

school vs. >High school CV1,

CV2, or CV3)

Maternal Marital Status M1

(Married or with Partner vs.

Single)

Insurance M1 (Medicaid vs. Other

or None)

Maternal Age M1

Child's age (months) CV1, CV2,

CV3

Birth: Gestational Age and Birth Weight-

for-Length %tile

At CV1, CV2, CV3

o Weight-for-length %tile CV1, CV2,

CV3

o Possible SE Problem (CV1, CV2)

(Yes vs. No)

o Total SE Problem CV1, CV2, CV3

o Cognitive Risk CV1

(competent vs. emerging or risk)

o Cognitive Class at CV2, CV3

(average and above vs. below

average)





Potential Predictors (cont’d)

Maternal characteristics TLEQ, M2

BSI, M2, CV1, CV2, CV3:

Anxiety, Depression, Global

Severity Index (GSI) T-scores

EPDS score (depression)CV1

Maternal BMI, M1

Temperament, M1:

Dysthymic, Cyclothymic,

Hyperthymic, and Irritable

Temperament Scores

WASI (intelligence) Total

Score

PSI, CV1, CV2, CV3

o Defensive Response;

Parental Distress; Parent-

Child Dysfunctional

Interaction; Difficult Child

%tiles; Total PSI %tile

CAPI, CV1, CV2, CV3

o Abuse, Distress, Rigidity,

Unhappiness, Problems

with Child and Self,

Problems with Family,

Problems with Others

scores





Demographics Mean Ln-Cortisol ng/mg at CV1

(similar differences at CV2, CV3)

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

Race Insurance Gender Marital Status Education

Me

an

Ln

- C

ort

iso

l n

g/

mg

at

CV

1

Bla

ck

Wh

ite

Oth

er

or

No

ne

Me

dic

aid

Male

Fe

ma

le

Sin

gle

Ma

rrie

d o

r L

ivin

g w

/

Pa

rtn

er

>H

S

<=

HS

P<0.0001 P<0.0001 P=0.1873 P=0.0001 P=0.0085





31.23 36.25 21.13 16.25 8.97 10.17 0

20

40

60

80

100

120

CV1 CV2 CV3

Me

an n

g/m

g Cortisol by Race

Black White

Mean hair cortisol was higher among Black vs. White children at

CV1 (p<0.0001), CV2 (p<0.0001), CV3 (p<0.0001)





Distribution of Cortisol at CV1 Cortisol (ng/mg) at CV1

(Black+White)

Ln-Transformed Cortisol at CV1

(Black+White)

0

50

100

150

200

250

300

350

0 73 145 218 290 363 435

Fre

qu

en

cy

Cortisol (ng/mg)

0

20

40

60

80

-3 -2 0 1 3 4 6

Fre

qu

en

cy

Ln- Cortisol (ng/mg)





Distribution of Cortisol at CV1

0

40

80

120

160

0 91 183 274 365

Fre

qu

en

cy

Cortisol (ng/mg)

0

20

40

60

80

-2 0 1 3 5

Fre

qu

en

cy

Ln- transformed Cortisol (ng/mg)

Black

0

40

80

120

160

0 115 230 345 More

Fre

qu

en

cy

Cortisol (ng/mg)

0

20

40

60

80

-3 -1 1 3 5

Fre

qu

en

cy

Ln-transformed Cortisol (ng/mg)

White





0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

CV1 CV2 CV3

Mean

Ln

-Co

rtis

ol n

g/m

g

Ln-Cortisol by Race

Black White

Mean hair Ln-cortisol was higher among Black vs. White children at CV1

(p<0.0001), CV2 (p<0.0001), CV3 (p<0.0001)





Linear Regression Model for CV1

Overall

R2 = 16%;

N=371

Variable Name Estimate SE P-value

Maternal Race [Black vs. White] 1.18592 0.15077 <0.0001

Gestational Age -0.08338 0.04723 0.0783

Depression T-score CV1 0.02621 0.01161 0.0246

Abuse Score CV1 -0.00282 0.00136 0.0391

Black

R2 = 3%;

N=214

Unhappiness Score CV1 -0.06050 0.0299 0.0443

Cognitive Risk CV1 [emerging

or at risk vs. competent] 0.43395 0.24622 0.0794

White

R2 = 12%;

N=158

Child's age (months) CV1 -0.22858 0.07827 0.0040

Gestational Age -0.17999 0.09146 0.0509

Weight-for-length CV1 %tile -0.00743 0.00390 0.0585

Parent-Child Dysfunctional

Interaction %tile CV1 0.00937 0.00419 0.0270






Overall

R2 = 18%;

N=282



Dysthymic Score M1 0.14455 0.07105 0.0429

EPDS Score CV1 0.04992 0.02594 0.0554

Maternal Age M1 -0.03314 0.01899 0.0820

Black

R2 = 6%;

N=121

Child's age in months CV2 0.18334 0.08785 0.0390

Maternal Insurance M1 [Medicaid

vs. other or none] 0.64361 0.33580 0.0577

White

R2 = 11%;

N=169

Maternal Adverse Events [> 5

events vs. < 5 events] 0.77781 0.31589 0.0148

Dysthymic Score M1 0.14799 0.08827 0.0955

EPDS Score CV1 0.08677 0.04192 0.0400

Total PSI CV2 -0.00880 0.00416 0.0362






Overall

R2 = 26%;

N=174



Birth weight-for-length %tile 0.00898 0.00390 0.0227

Anxiety T-score CV3 -0.02450 0.01220 0.0462

Black

R2 = 17%;

N=76

Maternal Adverse Child Events [Yes

vs. No] 0.60020 0.29057 0.0425

Depression T-score M2 -0.03430 0.01690 0.0461

Birth weight-for-length %tile 0.01300 0.00507 0.0125

White

R2 = 12%;

N=95

Total Parenting Stress %tile CV3 0.00849 0.00491 0.0873

Anxiety Tscore CV3 -0.06224 0.01766 0.0007





Linear Regression Models Ln-Cortisol

(ng/mg)

Black & White Black White

CV1

Mat Race [+]

Gestational Age [–]

Mat Depression CV1[+]

CAPI at CV1 [–]

R2 = 16%

Mat Unhappiness CV1[–]

Child at Cognitive Risk [+]

R2 = 3%

Child Age (mo.) [–]

Gestational Age [–]

Wt./Length %tile CV1 [–]

Parent-Child Dysfunction[+]

R2 = 12%

CV2

Mat Race [+]

Dysthymic Score [+]

Mat Depression CV1[+]

Maternal Age [–]

R2 = 18%

Child’s Age at CV2 [+]

Mat Insurance [+]

R2 = 6%

Mat Adverse Events >5 [+]

Dysthymic Score [+]

Mat Depression CV1 [+]

Total Stress (PSI) [+]

R2 = 11%

CV3

Mat Race [+]

Birth Wt./Length %tile [+]

T score Anxiety [–]

R2 = 26%

Mat Adverse Child Event[+]

Tscore Mat Depression[–]

Birth Wt./Length %tile [+]

R2 = 17%

Total Stress PSI [+]

Tscore Anxiety [–]

R2 = 12%





Conclusions The CANDLE birth cohort examines the impact of

early adversity on neurocognitive outcomes

Hair cortisol levels were significantly higher in Black

vs. White children suggesting dysregulation of the

HPA axis

Children having HPA axis dysregulation may be at

greater risk for poorer psychosocial, cognitive, and

clinical outcomes

Ongoing data collection and analyses will further

define the factors associated with toxic stress in

early life

Future research will include longitudinal analyses

when the CANDLE dataset is complete





• CANDLE Staff

Maureen Sorrells, MPH, Study Manager

Matt Hood, MS, Research Specialist

• Science Coordinator

Thuy Nguyen, MPH

• Data Analysts

Angela Moore, MPH, CPH

• Biostatisticians

Tamekia Jones, PhD

• PNL Research Students

Karmen McPhearson

Aaron Kala

Stephanie Allen-Winters CANDLE Participants:

parents and their children





To describe my mother would be to write about a hurricane in its perfect

power. Maya Angelou

Mother love is the fuel that enables a normal human being to do the

impossible. Marion C. Garretty

When you feel neglected, think of the female salmon, who lays 3,000,000

eggs but no one remembers her on Mother's Day. Sam Ewing

Stress is nothing more than a socially acceptable form of mental illness.

Richard Carlson

If you ask what is the single most important key to longevity, I would have

to say it is avoiding worry, stress and tension. And if you didn’t ask me,

I’d still have to say it. George F. Burns

Poets, Humorists, Athletes, and

Celebrities: Stress Perspective





APPENDIX SLIDES

The following slides are for reference and additional information.





Statistical Approach

• Model selection

• Stepwise regression

• Results for outcome Ln Hair Cortisol

• CV1: Overall, Black, White







Limitations

• Single platform for our measurements of cortisol

• Alpo detection

• No normative data exists for cortisol levels





Assay Specificity (Cross-Reactivity)

Steroid

% Cross

Reactivity

State

Presence

in Hair

Protein

Cortisol 100 Adrenal produced steroid hormone Yes

Prednisolone 13.6 Prescription drug, synthetic

glucocorticoid

?

Corticosterone 7.6 Adrenal precursor to aldosterone ?

Deoxycorticosterone 7.2 Adrenal precursor to aldosterone ?

Progesterone 7.2 Adrenal produced steroid hormone ?

Cortisone 6.2 Adrenal produced steroid hormone Yes

Deoxycortisol 5.6 Steroid that can be oxygenated to

cortisol

?

Pednisone 5.6 Prescription drug, synthetic

glucocorticoid

?

Dexamethasone 1.6 Prescription drug, synthetic

glucocorticoid

?





Physiological Functions of Cortisol

• Blood pressure maintenance

• Calcium absorption and bone formation

• Anti-inflammatory activity

• Increasing gluconeogenesis (generation of

blood sugar)

• Aiding in fat, protein and carbohydrate

metabolism

• Increases under stress; therefore is a

biomarker of changes in the HPA axis





ANOVA Total (Black + White) Black White

CV1 Predictor N Mean SD P-value N Mean SD P-value N Mean SD P-value

Maternal Race

Black 214 2.68 1.23 <.0001

White 159 1.60 1.48

Child's gender

female 207 2.13 1.38 0.1873

120 2.60 1.16 0.2855

87 1.48 1.40 0.2807

male 166 2.33 1.52 94 2.78 1.31 72 1.74 1.58

Maternal Education (CV1)

<=HS 163 2.44 1.32 0.0085

124 2.73 1.19 0.4618

39 1.52 1.28 0.7003

>HS 210 2.04 1.52 90 2.60 1.28 120 1.62 1.55

Maternal Marital Status (M1)

Married or LivingWithPartner 249 2.01 1.50 0.0001

102 2.64 1.25 0.6914

147 1.58 1.50 0.6042

Single 124 2.62 1.24 112 2.71 1.21 12 1.81 1.26

Insurance (M1)

Medicaid 180 2.53 1.18 <.0001

149 2.70 1.07 0.6255

31 1.71 1.33 0.6448

Other or None 193 1.92 1.60 65 2.61 1.54 128 1.57 1.52

Possible SE Problem (CV1)

No 298 2.15 1.45 0.0916

153 2.68 1.15 0.8965

145 1.59 1.52 0.9738

Yes 73 2.47 1.44 60 2.66 1.43 13 1.61 1.17

Cognitive Risk (regroup) (CV1)

competent 326 2.20 1.40 0.5102

185 2.63 1.20 0.1468

141 1.63 1.45 0.3978

emerging or at risk 47 2.35 1.71 29 2.98 1.37 18 1.32 1.73

Maternal Adverse Child Events (binary)

No 236 2.20 1.48 0.8010

120 2.74 1.21 0.3919

116 1.65 1.53 0.5061

Yes 137 2.24 1.39 94 2.59 1.25 43 1.47 1.37

Maternal Adverse Events (binary)

<5 events 301 2.21 1.47 0.7825

169 2.64 1.27 0.4601

132 1.65 1.53 0.3664

>=5 events 72 2.26 1.33 45 2.80 1.07 27 1.36 1.25

ANOVA: Ln-Cortisol at CV1





ANOVA: Ln-Cortisol at CV2 ANOVA

Total (Black + White) Black White


Maternal Race

Black 121 2.52 1.61 <.0001

White 183 1.12 1.54

Child's gender

Female 186 1.80 1.74 0.1190

89 2.62 1.58 0.2503

97 1.04 1.54 0.4816

Male 118 1.48 1.66 32 2.24 1.70 86 1.20 1.56


<= High School 103 1.91 1.74 0.0839

59 2.56 1.70 0.7863

44 1.04 1.38 0.7135

> High School 201 1.55 1.69 62 2.48 1.54 139 1.14 1.60


Married or LivingWithPartner 218 1.35 1.67 <.0001

46 2.25 1.77 0.1476

172 1.11 1.56 0.7884

Single 86 2.50 1.55 75 2.69 1.50 11 1.24 1.33

Insurance (M1)

Medicaid 125 2.28 1.63 <.0001

92 2.67 1.54 0.0702

33 1.18 1.35 0.7991

Other or None 179 1.26 1.65 29 2.05 1.76 150 1.10 1.59

Possible SE Problem (CV2)

NO 255 1.60 1.73 0.0813

87 2.56 1.65 0.6747

168 1.10 1.55 0.7105

Yes 49 2.07 1.61 34 2.42 1.54 15 1.26 1.51

Cognitive Class (regroup) (CV2)

Average and Above 283 1.65 1.69 0.2989

109 2.52 1.51 0.9963

174 1.10 1.57 0.5383

Below Average 21 2.05 1.98 12 2.52 2.43 9 1.43 0.98


No 211 1.58 1.67 0.1547

68 2.52 1.56 0.9662

143 1.14 1.54 0.7085

Yes 93 1.89 1.80 53 2.53 1.70 40 1.03 1.58


<5 events 241 1.56 1.73 0.0264

92 2.54 1.62 0.8505

149 0.96 1.51 0.0048

>=5 events 63 2.10 1.60 29 2.47 1.61 34 1.79 1.55





ANOVA: Ln-Cortisol at CV3 ANOVA

Total (Black + White) Black White


Maternal Race

Black 92 2.39 1.25 <.0001

White 96 0.82 1.46

Child's gender

Female 125 1.70 1.47 0.1714

72 2.41 1.233 0.7842

53 0.73 1.18 0.5288

Male 63 1.37 1.75 20 2.33 1.335 43 0.92 1.76


<= High School 78 1.97 1.27 0.0051

57 2.35 1.076 0.6745

21 0.93 1.18 0.6529

> High School 108 1.32 1.72 35 2.46 1.504 73 0.77 1.55


Married or LivingWithPartner 124 1.22 1.62 <.0001

32 2.39 1.447 0.9927

92 0.82 1.49 0.9195

Single 64 2.29 1.19 60 2.39 1.143 4 0.74 0.82

Insurance (M1)

Medicaid 96 2.09 1.37 <.0001

76 2.42 1.214 0.6805

20 0.85 1.24 0.9064

Other or None 92 1.06 1.60 16 2.28 1.444 76 0.81 1.52

Cognitive Class (regroup) (CV3)

Average and Above 167 1.52 1.59 0.1216

76 2.39 1.29 0.9290

91 0.80 1.45 0.7361

Below Average 21 2.09 1.36 16 2.42 1.069 5 1.03 1.77


No 128 1.47 1.60 0.1224

58 2.20 1.432 0.0548

70 0.86 1.47 0.6543

Yes 60 1.85 1.50 34 2.72 0.768 26 0.71 1.46


<5 events 156 1.59 1.55 0.9864

78 2.35 1.225 0.3880

78 0.83 1.48 0.8232

>=5 events 32 1.58 1.69 14 2.66 1.392 18 0.75 1.43




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09202013 CANDLE SLLS Hair Cortisol KJSA&CRR