Investigating Inflammatory Mediators

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Investigating Inflammatory Mediators

Specific findings from prior clinical work specifically investigating inflammatory mediators have demonstrated differential biomarker patterns in umbilical cord serum from infants stratified by preterm and CP status [1]. Of the

potential inflammation markers that differed between cases and controls, the markers were lower (based on medians) in the preterm CP cases relative to controls but were higher, relative to controls, in the term CP cases.

 

Sampling from a different compartment (plasma) and using a different approach, a study by Lin et al. (2010) comparing school-age children who were former preterm births reported higher cytokine responses (increased tumor

necrosis factor-alpha [TNF-α] plasma levels and greater mRNA levels of toll- like receptor four [TLR4]) among those children with CP who were preterm relative to controls (children born preterm with normal development) [9].

Another study of blood, using a serial approach (i.e., repeated measures) in over 900 preterm infants, documented elevated concentration values of myriad inflammatory- relevant mediators which were associated with different

risk profiles depending on the sampling day [10]. Using CSF from preterm infants with brain injury, Douglas- Escobar and Weiss (2012) documented combinations of biomarker concentration values that could be used to

 

inform clinical decision making [4]. Using blood from a sample of children with and without CP, Zareen (2020) [11] found significantly increased levels of erythropoie- tin at baseline in children with CP compared with chil- dren in

the comparison group. In response to challenge (lipopolysaccharide), both groups had appropriate and comparable response profiles for interleukin-8, vascular endothelial growth factor (VEGF), TNF-α, and granu- locyte–

macrophage colony-stimulating factor (GM- CSF) levels. The children with CP showed a statistically significant lipopolysaccharide hypo-responsiveness profile for interleukin-1a, interleukin-1b, interleukin-2, and interleukin-6

levels. Collectively, the work to date consistently shows immune and inflammatory differ- ences in children with CP.

 

There remain gaps in our knowledge about the spe- cific linkages among various immune and related mechanisms driving hypothesized persistent inflamma- tory states in children with CP, and the relation among the various

biomarkers, risk factors, and specific out- comes. The overall generality of the findings to date for the CP population is limited by two kinds of problems, namely the relative difficulties in establishing valid pre- clinical models for

this purpose [12] and the extreme paucity of clinically-relevant biomarker research within this high-need vulnerable patient group. For example, the relation among the CSF biomarkers investigated by Douglas-Escobar and Weiss

(2012) and CP as an outcome was not clear [4]. More biomarker data of a comparable kind from the same and different compart- ments across the same and different age groups are needed. To the best of our knowledge, no

comparable work has investigated inflammatory-relevant molecu- lar biomarkers in CSF from children, adolescents, and young adults with CP.

 

The purpose of this preliminary investigation was exploratory. The design was cross-sectional using a single time point for specimen collection from a clini- cal sample. There were two specific aims. The first aim was to

document levels of inflammatory and related molecules in CSF in a sample primarily of school-age children with CP. To do so, for detectable analytes, participants were arrayed along each analyte’s con- centration gradient. The

second aim was to examine clinically relevant grouping variables (e.g., CP sever- ity, term/preterm birth) to identify any potentially rel- evant correlational patterns among the molecules. Our intent was to extend the work initiated

by Kaukola and Lin (described above) using a clinical sample in which standard-of-care surgical interventions were leveraged to gain access to CSF for future hypothesis-generating research purposes.

 

Page 3 of 13Goracke‑Postle et al. BMC Neurol (2021) 21:384

 

Methods Protocol approval This study was approved by the Institutional Review Board (IRB, #0809M46301) of the University of Minne- sota. Written informed consent was obtained from each participant or legal representative

(i.e., parent/guardian).

 

Participants This study utilized a single-time point cross-sectional design. Twenty-eight individuals (82% male) with CP participated (mean age = 9.74  years, SD = 4.36; range = 4–23). Specific CP diagnoses included: quad-

riplegia (n = 17), diplegia (n = 5), and triplegia (n = 5). Participants were included in the study if they (a) had cerebral palsy, (b) were between 3–25  years of age, and (c) were scheduled for initial intrathecal baclofen (ITB) pump

implant. Individuals were excluded if (a) they had an existing cerebral shunt; or (b) they had compounded dosing (i.e., opioid adjunctive to baclofen) through their pump. The participants were already characterized clini- cally

using the Gross Motor Functional Classification System for Cerebral Palsy (GMFCS) to categorize gross motor function. The GMFCS is a 5-level classification system based on self-initiated movement with emphasis on truncal

control and walking. The GMFCS is widely used clinically and in classifying individuals with CP for research studies [13].

 

For the subgroup comparisons, the breakdown of par- ticipant demographics was as follows: males (n = 23) and females (n = 5); non-quadriplegia (n = 11) and quad- riplegia (n = 17); Caucasian (n = 26) and other (n = 2); spastic

CP (n = 13) and mixed tone CP (n = 12); term birth, defined as 37 weeks or later (n = 6), preterm birth, defined as 28–37  weeks (n = 12) and extremely preterm birth, defined as less than 28 weeks (n = 9); seizure (n = 6) and no

seizure (n = 16) (Table 1).

 

CSF collection Patients were consented in accordance with an approved IRB protocol. If consent was given, CSF was collected during a standard-of-care surgical procedure (ITB pump implant). In all cases, the surgery proceeded

as usual until the spinal catheter had been placed. Then, the neurosur- geon collected 10–20 ml of CSF from the spinal catheter placed well above the spinal puncture site. This method avoided contaminating the collected CSF

with blood.

 

Immediately following collection, the CSF was placed on wet ice (+ 4  °C) and transported to a cold room for processing, centrifuged at 3000  rpm × 5  min, pipetted into 100 and 250 µL aliquots, flash frozen in liquid nitro- gen

and archived at -80  °C. After specimen collection,

 

the patient was monitored closely following routine operative and post-operative procedures. There were no adverse events.

 

CSF analyte analysis CSF was analyzed using conventional biochemical meth- ods based on commercially available enzyme-linked immunosorbent assay (ELISA) kits and expression lev- els of each marker were quantified.

Specifically, samples were tested by the Cytokine Reference Laboratory (CRL, University of Minnesota). This is a CLIA’88-licensed facility (license #24D0931212). Samples were analyzed for adrenocorticotropic hormone (ACTH),

agouti- related peptide (AgRP), brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), follicle-stimulating hormone (FSH), growth hormone (GH), luteinizing hormone (LH), prolactin (PRL), and thyroid

stimulating hormone (TSH) using the “Human Brain-Derived Protein Panel” on the Luminex platform and done as a multi-plex (Luminex instrument—Bioplex 100 [Bio-Rad, 1000 Alfred Nobel Drive, Hercules, CA, 94547], Software:

bio-plex Manager 4.0). The polysty- rene bead set (cat. # HPT-66  K-09) with kit lot number 1757143 was used. Kits/reagents were purchased from EMD Millipore Corporation, Billerica, MA. Interferon α2 (IFNα2), interleukin-1α (IL-

1α), interleukin-1ra (IL-1ra), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-10 (IL-10), interleukin-12p40 (IL-12p40), interleukin-12p70 (IL-12p70), interferon gamma-induced protein 10 (IP- 10), monocyte chemotactic protein

(MCP-1), mac- rophage inflammatory protein 1β (MIP1β), regulated on activation normal T expressed and secreted (RANTES), and tumor necrosis factorα (TNFα) were analyzed using the “Cytokine/Chemokine Panel 1” on the

Luminex plat- form and done as a multi-plex (Luminex instrument— Bioplex 100 [Bio-Rad, 1000 Alfred Nobel Drive, Hercules, CA, 94547], Software: bio-plex Manager 4.0). The poly- styrene bead set (cat. # MPXHCYTO-60  K-14)

with kit lot number 1757142 was used. Kits/reagents were pur- chased from EMD Millipore Corporation, Billerica, MA. Dynorphin A, neuropeptide Y, somatostatin, β endor- phin, cortisol, neurotensin, orexin A, substance P, mela-

tonin, oxytocin, and melanocyte-stimulating hormone (α-MSH) were analyzed using the “Human Neuropeptide Panel” on the Luminex platform and done as a multi-plex (Luminex instrument—Bioplex 100 Bio-Rad, 1000 Alfred

Nobel Drive, Hercules, CA, 94547], Software: bio-plex Manager 4.0). The polystyrene bead set (cat. # HNP- 35 K-08) with kit lot number 1823005 was used. Kits/rea- gents were purchased from EMD Millipore Corporation, Billerica,

MA.

 

Samples were assayed according to manufacturer’s instructions. ELISA employ the quantitative sandwich

 

Page 4 of 13Goracke‑Postle et al. BMC Neurol (2021) 21:384

 

enzyme immunoassay technique. The absorbance is measured on the microtiter plate reader (Bio-Rad model 550). The intensity of the color formed is proportional to the concentration of the sample. Fluorescent color- coded

beads coated with a specific capture antibody were added to each sample. After incubation, and washing, biotinylated detection antibody was added, followed by phycoerythrin-conjugated streptavidin. The beads were read on a

Luminex instrument (Bioplex 100) which is a dual-laser fluidics-based instrument. One laser deter- mines the analyte being detected via the color coding; the other measures the magnitude of the PE signal from the

 

detection antibody which is proportional to the amount of analyte bound to the bead. Samples were tested in duplicate and values were interpolated from 5 parameter- fitted (5PL) standard curves.

 

Statistical analyses Data analysis was exploratory and relied on visual analy- sis, descriptive statistics, and correlational analyses. First, visual analysis of each analyte was conducted to under- stand its distributional form and to

identify potential outliers. Further, measures of central tendency (means,

 

Table 1 Participant health information; M ± SD or n (%)

 

Note: gestational age was not available for one participant; Term birth = born 37 weeks gestation or later; preterm birth = born at 28–37 weeks gestation; extremely preterm birth = born at less than 28 weeks gestation; CP

Cerebral Palsy, GMFCS Gross Motor Function Classification System, level I ambulant without assistance, level II ambulant without assistive devices, limitations outside the home, level III ambulant with assistive devices,

wheelchair required outside the home, level IV non‑ ambulatory, self‑mobile in wheelchair with limitations, level V non‑ambulatory, self‑mobility very limited

 

Complete sample (n = 28)

 

Term birth (n = 6)

 

Preterm birth (n = 12)

 

Extremely preterm birth (n = 9)

 

Male 23 (82.1) 3 (50.0) 11 (91.7) 9 (100)

 

Ethnicity

 

Caucasian 26 (92.9) 6 (100.0) 12 (100.0) 8 (88.9)

 

African American 1 (3.6) 0 (0) 0 (0) 1 (11.1)

 

Other (not specified) 1 (3.6) 0 (0) 0 (0) 0 (0)

 

Epilepsy

 

None 16 (57.1) 3 (50.0) 7 (58.3) 6 (66.7)

 

History of seizure/diagnosis of epilepsy 6 (21.4) 1 (16.7) 4 (33.3) 1 (11.1)

Questionable seizure activity 3 (10.7) 1 (16.7) 0 (0) 1 (11.1)

 

Missing 3 (10.7) 1 (16.7) 1 (8.3) 1 (11.1)

 

CP topography

 

Hemiplegia 1 (3.6) 0 (0) 1 (8.3) 0 (0)

 

Diplegia 5 (17.9) 2 (33.3) 3 (25.0) 0 (0)

 

Triplegia 5 (17.9) 1 (16.7) 0 (0) 4 (44.4)

 

Quadriplegia 17 (60.7) 3 (50.0) 8 (66.7) 5 (55.6)

 

GMFCS

 

Level I 3 (10.7) 0 (0) 3 (25.0) 0 (0)

 

Level II 3 (10.7) 1 (16.7) 0 (0) 2 (22.2)

 

Level III 7 (25.0) 3 (50.0) 2 (16.7) 2 (22.2)

 

Level IV 8 (28.6) 1 (16.7) 4 (33.3) 3 (33.3)

 

Level V 6 (21.4) 1 (16.7) 2 (16.7) 2 (22.2)

 

Missing 1 (3.6) 0 (0) 1 (8.3)

 

Tone

 

Spastic 13 (46.4) 4 (66.7) 5 (41.7) 4 (44.4)

 

Mixed tone 12 (42.9) 2 (33.3) 5 (41.7) 4 (44.4)

 

Missing 3 (10.7) 0 (0) 2 (16.7) 1 (11.1)

 

Current feeding tube

 

Yes 8 (28.6) 2 (33.3) 2 (16.7) 4 (44.4)

 

No 19 (67.9) 4 (66.7) 10 (83.3) 5 (55.5)

 

Missing 1 (3.6) 0(0) 0 (0) 0 (0)

 

Days in NICU at birth Range of NICU days

 

76.25 ± 67.04 (0–300)

 

3.50 ± 7.00 (0–14)

 

52.73 ± 29.72 (10–120)

 

137.33 ± 64.76 (90–300)

 

Page 5 of 13Goracke‑Postle et al. BMC Neurol (2021) 21:384

 

medians) and variation (standard deviations, coefficients of variation) were calculated for each analyte.

 

Second, to understand the associations between ana- lytes a series of pairwise scatterplots and Pearson prod- uct-moment correlations were computed between each possible pair of analytes for the entire sample. The corre-

lations were tested for statistical significance against the null hypothesis of r = 0. Parallel analyses and plots were generated for the data set with missing data imputed with the lower limit / sensitivity number. Given the large

number of correlations tested (528), Type 1 errors were controlled for by using the false discovery rate correction discussed by Benjamini & Hochberg (1995). Correlations were considered against an alpha = 0.05, after the false

discovery rate correction was applied.

 

Third, to understand how the associations between analytes varied by subgroups, the correlational analyses described above were repeated for each of the gestational term subgroups. Given the large number of compari- sons

involved in these analyses, an alpha = 0.001 was set

 

for each test after the false discovery correction rate was applied.

 

Finally, differences in correlations between subgroups were also tested. To be included in between-group analysis, each correlation first had to be statistically significant within the subgroups. Thirty-two pairs of correlations were

statistically significant across all sub- groups. Then the identified correlations were tested against one another to check whether they were sig- nificantly different by birth status. To do this, Fisher’s r to z transformation was used

to calculate the differ- ence in correlations that met the criteria for inclusion, and tested for statistical significance of that difference; p ≤ 0.05.

 

Results CSF analyte expression in CP subjects Detectable CSF analytes were broken down, broadly, by the following categories: hormonal/endocrine brain-derived peptides or proteins: ACTH, AgRP,

 

Fig. 1 Visual representation of the direction and strength of the Pearson’s correlation coefficients between all 33 analytes assayed. Positive (blue), negative (red), strong (dark shading), and weak (light shading) correlations are

depicted

 

Investigating Inflammatory Mediators

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