. 2021 Aug 11;18(2):474–522. doi: 10.1007/s12015-021-10213-y

Table 3.

Effects of immune-related treatments in the rodent model of neonatal HI

Reference	Species and Model of HI	Immune-related treatment	Degree of neuroprotection^†, neuroinflammation, conclusion(s)
Martin D et al., 1994 PMID: 7867766	Sprague-Dawley rat P7 Left CCA ligation 120 min 7.5% O₂ Sacrifice time-point: 7 and 14 d (P14 and P21)	Recombinant human interleukin-1 receptor antagonist (rhIL-1ra, anakinra) Route: Repeated subcutaneous (s.c.) administration Dose: 100 mg/kg Treatment protocol: In the acute phase post-HI 4 groups of rat neonates receiving different dosing frequencies prior to and/or after the HI insult (up till 8 s.c. injections) - Group 1: 1 h prior hypoxia and at 0, 1, 3, 5, 9, 20 and 28 h - Group 2: at onset of hypoxia and at 1, 3, 5, 9, 20 and 28 h - Group 3: 1 h post hypoxia, and then 3, 5, 9, 20 and 28 h - Group 4: 3 h post hypoxia, and then 5, 7, 20 and 28 h In clinical use for other conditions: yes, e.g. for gout	Degree of neuroprotection 7 d post-HI, assessment of right and left hemisphere dry weights disparities: - Group 1: ~55% neuroprotection (). - Group 2: ~95% neuroprotection (). - Group 3: ~80% neuroprotection (). - Group 4: ~54% neuroprotection (NS). 14 d post-HI, evaluation of loss of striatal neurons in cresyl violet stained brain slices only in group 1: - ~65% neuroprotection (). Cerebral inflammation post-treatment Not examined Conclusion IL-1 receptor activation plays a role in the pathogenesis of neonatal HI brain injury.
Hagberg H et al., 1996 PMID: 8888290	Wistar rat P7 Left CCA ligation 70–100 min 7.70% O₂ Sacrifice time-points: 1, 3, 6, 10, 20, and 48 h and 14 d (P21)	rhIL-1ra Route: Intracerebral administration in the ipsilateral hemisphere Dose: 3.3 or 5 μg/rat pup Treatment protocol: Prophylactic, or in the acute phase post-HI Administration either before (3.3 μg/rat pup) or immediately after HI (5 μg/rat pup) In clinical use for other conditions: see above	Degree of neuroprotection 14 d post-HI, assessment of right and left hemisphere dry weights disparities: - HI duration of 100 min and IL-1ra pretreatment: ~24% neuroprotection (*) - HI duration of 70 min and IL-1ra post-treatment: no change in mean injury Cerebral inflammation post-treatment* Not examined Conclusion rhIL-1ra mediated neuroprotection, despite being moderate, suggests the pathophysiological significance of IL-1 in neonatal HI.
Liu X et al., 1996 PMID: 8947953	Sprague-Dawley rat P7 Right CCA ligation 150–195 min 8% O₂ Sacrifice time-point: 5 d (P12)	Platelet-activating factor (PAF) antagonist BN52021 Route: intraperitoneal (i.p.) injection 3 treatment protocols: Prophylactic, or in the acute phase post-HI - Pretreatment: 2 serial injections (25 mg/kg/injection), 1 immediately before the onset of hypoxia, and a second one 1 h after the end of hypoxia - Post-treatment: 2 serial injections (25 mg/kg/injection), 1 immediately after the end of hypoxia, and then 2 h later - Post-treatment-dose effect: 2 injections, immediately and 2 h after the end of hypoxia- different doses tested (12.5, 25 or 50 mg/kg/injection) In clinical use for other conditions: PAF antagonists are a heterogenous group of medication, some of which are used for different diseases	Degree of neuroprotection 5 d post-HI, assessed on cresyl violet stained brain slices: - Incidence of infarction 90% in vehicle-treated rats vs 33% in BN52021-pretreated rats (*) and 30% in BN52021-post-treated rats (). - Neuroprotection per region • Pretreatment: 82% in cortex, 74% in striatum, 90% in hippocampus. • Post-treatment: 72% in cortex, 46% in striatum, 73% in hippocampus. 5 d post-HI, assessed by interhemispheric weight differences for the post-treatment dose-effect study: 18.9% (NS), 44.8% () and 32.5% () neuroprotection observed in rats treated with 12.5, 25 or 50 mg/kg/dose compared with controls; differences in neuroprotection among the three doses not statistically different. Cerebral inflammation post-treatment Not examined Conclusion PAF appears to be one of the acute inflammatory factor mediating perinatal HI brain injury; PAF antagonists may represent a pharmacologic approach to attenuate the severity of HI-brain injury.
Hudome S et al., 1997 PMID: 9128280	Wistar rat P7 Right CCA ligation 135 min 8% O₂ Sacrifice time-points: 0, 5, and 30 min, and 2, 4, 8, 18, and 42 h	Anti-rat neutrophil serum (ANS) compared to allopurinol Note: rabbit polyclonal anti-rat neutrophil serum (ANS) is used to deplete neutrophils; comparison to allopurinol, a drug that reduces neutrophil accumulation, and a known neuroprotective agent Route: i.p. for ANS and s.c. for allopurinol Dose: Allopurinol 135 mg/kg Treatment protocol: In the acute phase post-HI - 4 treatment groups: allopurinol, neutropenic, both and none - ANS is injected i.p. at time of surgery - Allopurinol is injected 15 min post-hypoxia In clinical use for other conditions: no, ANS is experimental only	Degree of neuroprotection 42 h post-HI, assessed by quantifying brain swelling (wet vs dry weight of posterolateral half of each cerebral hemisphere): - ~70% (*) reduction in brain swelling by ANS-induced neutropenia and allopurinol. The application of both treatments confers no synergistic effect. Cerebral inflammation post-treatment* Not examined. Conclusion by study authors Neutropenia before the insult is neuroprotective, thus neutrophils may play a role in the pathogenesis of neonatal HI, although the mechanisms remain unclear.
Arvin K et al., 2002 PMID: 12112047	Sprague-Dawley rat P7 Left CCA ligation 150 min 8% O₂ Sacrifice time-points: 24 h and 1 w	Minocycline Route: i.p. Dose: 45 mg/kg Treatment protocol: Prophylactic, or in the acute phase post-HI Single injection immediately before placement in hypoxic chamber, or immediately after removal from hypoxic chamber In clinical use for other conditions: yes, for various infections	Degree of neuroprotection 7 d post-HI, measurement of the amount of surviving tissue in cresyl violet stained brain slices, and calculation of % tissue loss in HI-IL vs HI-CL hemisphere: - Minocycline 45 mg/kg before hypoxia: 72% (**), 100% () and 85% (**) protection in striatum, hippocampus, and cortex. - Minocycline 22.5 mg/kg before hypoxia: 61% (NS), 72% (NS) and 71% () protection in striatum, hippocampus, and cortex. - Minocycline 45 mg/kg immediately after hypoxia: similar results as when given before hypoxia. - Minocycline 45 mg/kg 3 h after hypoxia: 28% (NS), 9% (NS) and 22% (NS) protection in striatum, hippocampus, and cortex. Cerebral inflammation post-treatment Not examined Conclusion Minocycline at 45 mg/kg provides near complete neuroprotection after neonatal HI only if given immediately before or after HI. Administration 3 h post-HI is not neuroprotective.
Palmer C et al., 2004 PMID: 14739365	Wistar rat P7 Right CCA ligation 135 min 8% O₂ Sacrifice time-points: - 42 h post-HI for brain swelling - 14 d post-HI for brain atrophy - 0 and 6 h post-HI for analysis of brain energy metabolites	Anti-rat neutrophil serum (ANS) Route: s.c. Treatment protocol: Prophylactic, or in the acute phase post-HI - before HI group: immediately after carotid ligation - after HI group: 4–8 h after hypoxia - 2 corresponding control groups (HI animals exposed to normal rabbit serum) In clinical use for other conditions: no, experimental only	Degree of neuroprotection 42 h post-HI, measurement of brain swelling: - In the before HI group, neutropenia reduces brain swelling by 75% (**). - In the after HI group, no reduction in brain swelling. - 14 d post-HI, measurement of brain atrophy: - In the before HI group, neutropenia reduces IL hemisphere atrophy by 61% (). - In the after HI group, neutropenia is not protective. Cerebral inflammation post-treatment Not examined Conclusion Neutrophils play a role in early HI-induced neuropathogenesis. Anti-neutrophil strategies should be applied very early after HI to be neuroprotective.
Jantzie L et al., 2005 PMID: 15647741	Sprague-Dawley rat P7 Right CCA ligation 150 min 8% O₂ Sacrifice time-points: - 1 w (P14) for brain stainings - 1, 2, and 4 h for immunoblotting	Doxycycline (DOXY) Route: i.p. Dose: 10 mg/kg Treatment protocol: Prophylactic, or in the acute phase post-HI Injection of DOXY as a one-time dose - Immediately before HI - 1 h after HI - 2 h after HI - 3 h after HI In clinical use for other conditions: yes, for various infections Can cross BBB	Degree of neuroprotection 7 d post-HI, assessment of NeuN immunoreactivity: In CA1 hippocampus, the average cell loss is ~30% in control animals, whereas it is ~10% in animals treated with DOXY, regardless of injection time-points, which means a ~ 70% neuroprotection. Cerebral inflammation post-treatment At 7 d post-HI, trend towards a reduction in the number of ED-1⁺ cells in the IL hippocampus, thalamus and cortex, with a greater extent when DOXY is administered before HI. Conclusion DOXY protects the neonatal brain from HI when applied in a short time window post-HI.
Sun Y et al., 2005 PMID: 16040592	Sprague-Dawley rat P7 Right CCA ligation 150 min 8% O₂ Sacrifice time-points: 3, 7, 14, and 21 d	recombinant human Erythropoietin (rh-EPO)^§ Route: i.p. Dose: 5 U/g body weight Treatment protocol: In the acute and subacute phase post-HI 3 injections, namely injection at 24 h post-HI and for 2 additional days In clinical use for other conditions: yes, and in clinical trials for infants with CP (NCT# 02811263) Can cross a dysfunctional blood-brain barrier	Degree of neuroprotection 7, 14 and 21 d post-HI, assessed by measurement of IL and CL wet weight disparities: ~47–77% (**) protection at these 3 time-points. Cerebral inflammation post-treatment* - rh-EPO abolishes HI-induced rise in IL-1β protein levels in the ipsilateral hemisphere 3, 7, and 14 d post-injury, but does not affect mRNA levels of TNF-α between 3 and 14 d post-HI. - It limits HI-induced leukocyte infiltration in the parieto-occipital cortex, observed with CD4 and CD68 IHC stainings. Conclusion 3 doses of rh-EPO is neuroprotective after HI. A potential mechanism of neuroprotection may be through reducing HI-associated elevation in IL-1β and through limiting leukocyte infiltration.
Nijboer C et al., 2008 PMID: 18420952	Wistar rat P7 Right CCA ligation 120 min 8% O₂ Sacrifice time-points: 1 h, 3 h, 6 h, 12 h, 48 h and 6 w	TAT-NBD, IKK/NF-κB inhibitor - TAT-NBD: peptide inhibitor of the IKK complex, the NEMO Binding Domain (NBD)-peptide coupled to the protein transduction sequence of HIV-TAT to facilitate cerebral uptake - mutant TAT-NBD - biotinylated TAT-NBD Route: i.p. Dose: 20 mg/kg Treatment protocol: In the acute phase post-HI administration at various time points post-HI: 0, 3, 6, 9 and 12 h after HI In clinical use for other conditions: no specific NF-κB inhibitor in use yet, but unrelated drugs may target this signaling pathway Can cross BBB	Degree of neuroprotection 48 h and 6 w post-HI, assessed by MAP2 staining and hematoxylin-eosin respectively: - ~40–56% (*) protection at 48 h post-HI if TAT-NBD administered 0/3 h after HI, ~35% () if administered 6 h after HI. - ~75% (**) protection at 6 w post-HI if administered 0/3 h after HI. - Administration of TAT-NBD at 9 or 12 h post-HI is not neuroprotective. Cerebral inflammation post-treatment* TAT-NBD treatment immediately after HI does not induce any changes in the HI-induced increase in mRNA levels of cytokine (TNF-α, IL-1β, IL-4, IL-10 and IL-1RA) at 3 h post-HI, even though NF-κB activity is fully inhibited at this time point. Conclusion Inhibition of the cerebral IKK/NF-kB pathway early after HI is neuroprotective, but does not involve abrogation of early cytokine expression. This early cytokine induction by neonatal HI may contribute to neuroprotection.
Jin Y et al., 2009 PMID: 19520991	Wistar rat P7 Right CCA ligation 75 min 8% O₂ Sacrifice time-points: 0, 1 h, 2 h, 4 h, 24 h, 48 h and 4 w	cromolyn, mast cell stabilizer Route: s.c. Dose: 50 mg/kg body weight Treatment protocol: In the acute phase post-HI 3 injections, i.e. immediately after HI, and 1 and 24 h post-HI In clinical use for other conditions: yes, for asthma	Degree of neuroprotection 1, 2 and 4 w post-HI, scoring of Fluoro-Jade B stainings, and measurement of IL/CL hemisphere ratio on H&E stained brain slices: - 61-75% () improvement in score of brain damage at the time-points examined. - Cavitation is absent in all cromolyn treated rats, whereas it is present in 30% and 50% of vehicle-treated animals at 2 and 4 w post-HI, respectively. - IL/CL ratio restored to normal in cromolyn treated rats (). Cerebral inflammation post-treatment Astrocytic and microglial activation, scored with GFAP and OX-42 antibodies respectively, are significantly reduced at the time points examined. Conclusion Mast cells appear to contribute to the inflammatory response to neonatal HI and could be the initiators of the immune response.
Nijboer C et al., 2009 PMID: 19628795	Wistar rat P7 Right CCA ligation 120 min 8% O₂ Sacrifice time-points: 3, 6, 24 and 48 h	TAT-NBD, IKK/NF-κB inhibitor; TAT-JBD, JNK inhibitor; and etanercept, TNF-α inhibitor TAT-NBD, mutant TAT-NBD or TAT-JBD Route: i.p. Dose: 20 mg/kg for TAT-NBD peptides and 10 mg/kg for TAT-JBD Treatment protocol: administered 0 and 3 h after HI Etanercept (a fusion protein of TNF-R2 and human IgG₁): Route: i.p. Dose: 5 mg/kg Treatment protocol: In the acute phase post-HI administered directly after HI alone or with TAT-NBD In clinical use for other conditions: etanercept is used for treating some autoimmune diseases (e.g. rheumatoid arthritis); the other compounds are experimental only	Degree of neuroprotection 48 h post-HI, measurement of CL and IL areas on MAP2 stained brain slices: - NBD: 75–88% (**). -JBD: 25% (). - NBD + JBD: 50% (**). - Etanercept: 25% (). - NBD + Etanercept: 44% (* vs NBD). Cerebral inflammation post-treatment - Combined NF-κB inhibition and JNK/AP-1 prevents HI-induced TNF-α production and reduces neuroprotection. - Etanercept alone has a significant neuroprotective effect, but not as great as that provided by NF-κB inhibition. - Etanercept treatment reduces the protective effect of TAT-NBD. Conclusion JNK/AP-1 inhibition and TNF-α inhibition reduce the neuroprotective effect of NF-κB inhibition: after HI is mediated in part by JNK/AP-1 activity and TNF-α production.
Fathali N et al., 2010 PMID: 20029340	Sprague-Dawley rat P10 Right CCA ligation 120 min 8% O₂ Sacrifice time-points: 72 h, 2 w, or 6 w	NS398, selective cyclooxygenase-2 (COX-2) inhibitor Route: i.p. Dose: 2 dosage regimens, either 10 mg/kg (NS-10 group), or 30 mg/kg (NS-30 group) Treatment protocol: In the acute and subacute phase post-HI six injections (1, 6, 24, 36, 48, and 60 h) after hypoxia In clinical use for other conditions: going out of clinic due to adverse cardiac events	Degree of neuroprotection 2 and 6 w post-HI, measurement of IL/CL weight ratio: - NS-10 group: 63% at 2 w () and 57% () at 6 w. - NS-30 group: 79% at 2 w () and 75% () at 6 w. 6 w post-HI, behavior analysis: improvement in, or restoration of many HI-induced neurobehavioral deficits in NS-10 and NS-30 treated animals. Cerebral inflammation post-treatment - COX-2 immunopositive signals (western blotting and IHC staining of ipsilateral brain) are significantly reduced in NS398 treated HI animals in comparison to HI-vehicle animals. - At 72 h post-HI, IL-6 staining and concentration in ipsilateral brain is increased in HI-vehicle exposed rats, and significantly reduced by NS-30. Qualitative evaluation of single immunofluorescent staining for Iba1, CD68 and MPO in ipsilateral brain indicates that NS30 attenuates significantly HI-induced increase in these markers. Conclusion COX-2 inhibition is neuroprotective; effects may be mediated by anti-inflammatory actions, through IL-6 reduction, decreased activation of microglia and of infiltration of macrophages and neutrophils.
Cikla U, 2016 PMID: 26857490	C57BL/6 J mice - P9 “P9 neonatal mice/brain” - P30 “P30 juvenile mice/brain” Left CCA ligation 50 min 10% O₂ Sacrifice time-points: 2, 9 and 60 d	Minocycline Route: i.p. Dose: 40 mg/kg Treatment protocol: In the acute phase post-HI Two injections, one at 2 h and the second one at 24 h post-HI In clinical use for other conditions: see above	Degree of neuroprotection 2 and 9 d post-HI, scoring of damage on MAP2 stained brain slices: - P9 neonatal brain: 73% () and 87% () protection at 2 and 9 d post-HI. - P30 juvenile brain: no protection at both days; of note, HI-induced brain damage less severe at P30 than at P9. Brain atrophy at 9 and 60 d post-HI by T2-weighted MRI: - P9 neonatal brain: no significant impact on volume loss at both days. - P30 juvenile brain: no significant impact on volume loss at 9 d, but a slight impact at 60 d. Hippocampal learning by Morris water maze test: - P9 neonatal brain: no amelioration in HI-induced learning deficits. - P30 juvenile brain: significant improvement. Cerebral inflammation post-treatment Impact of minocycline on total counts of microglia (CD11b⁺/CD45⁺) - Significantly suppresses HI-induced increase in microglia counts in the IL hippocampus from P9 and P30 brains 2 and 9 d post-HI; this is also observed in the cortex and striatum from P9 brains, but less in the same regions from P30 brains. Impact of minocycline on percentage of activated microglia (CD45^+med/total microglia) - 2 d post-HI, in the IL hippocampus, induces ~70% reduction in the proportion of activated microglia in both neonatal and juvenile brains. - 9 d post-HI in the IL cortex and striatum - P9 neonatal brain: significant decrease in the percentage of activated microglia - P30 juvenile brain: no impact of minocycline on HI-induced late microglia activation Conclusion Minocycline has different neurological outcome depending on the age at which HI-injury is induced. Early improvements in neurologic injury do not necessarily predict long-term improvements in neurologic function.
Herz J et al., 2018 PMID: 30127782	C57BL/6 J mice P9 Right CCA ligation 60 min 10% O₂ Sacrifice time-points: 1 w (P16)	FTY720 (Fingolimod), a sphingosin-1-phosphate analog that reduces peripheral lymphocytes Route: i.p. Dose: 1 mg/kg body weight Treatment protocol: In the acute phase post-HI single injection within 20 min after hypoxia In clinical use for other conditions: yes, for multiple sclerosis	Degree of neuroprotection 1 w post-HI, scoring of injury in cresyl violet stained brain slices: - Significant worsening of neuropathology in FTY720-treated animals versus saline-exposed mice. Cerebral inflammation post-treatment - FTY720 treatment induces a slight but not significant increase in Iba1 protein levels (assessed by western blot). - Despite FTY720 induced depletion of circulating T cells, the total cerebral leukocyte infiltration assessed qualitatively by IHC is unaffected by FTY720. This is explained by an increased infiltration of innate immune cells, mainly neutrophils and inflammatory macrophages. Conclusion Pharmacological mediated T cell depletion increases HI-induced brain injury; this contrasts with the neuroprotective effect of FTY720 observed in adult models of stroke. Thus, neonatal T cells may promote endogenous neuroprotection.

Reference

Species and Model of HI

Immune-related treatment

Degree of neuroprotection^†, neuroinflammation, conclusion(s)

Martin D et al., 1994

PMID: 7867766

Sprague-Dawley rat

Left CCA ligation

120 min 7.5% O₂

Sacrifice time-point:

7 and 14 d (P14 and P21)

Recombinant human interleukin-1 receptor antagonist (rhIL-1ra, anakinra)

Route: Repeated subcutaneous (s.c.) administration

Dose: 100 mg/kg

Treatment protocol:

In the acute phase post-HI

4 groups of rat neonates receiving different dosing frequencies prior to and/or after the HI insult (up till 8 s.c. injections)

- Group 1: 1 h prior hypoxia and at 0, 1, 3, 5, 9, 20 and 28 h

- Group 2: at onset of hypoxia and at 1, 3, 5, 9, 20 and 28 h

- Group 3: 1 h post hypoxia, and then 3, 5, 9, 20 and 28 h

- Group 4: 3 h post hypoxia, and then 5, 7, 20 and 28 h

In clinical use for other conditions: yes, e.g. for gout

Degree of neuroprotection

7 d post-HI, assessment of right and left hemisphere dry weights disparities:

- Group 1: ~55% neuroprotection (*).

- Group 2: ~95% neuroprotection (**).

- Group 3: ~80% neuroprotection (**).

- Group 4: ~54% neuroprotection (NS).

14 d post-HI, evaluation of loss of striatal neurons in cresyl violet stained brain slices only in group 1:

- ~65% neuroprotection (*).

Cerebral inflammation post-treatment

Not examined

Conclusion

IL-1 receptor activation plays a role in the pathogenesis of neonatal HI brain injury.

Hagberg H et al., 1996

PMID: 8888290

Wistar rat

Left CCA ligation

70–100 min 7.70% O₂

Sacrifice time-points:

1, 3, 6, 10, 20, and 48 h and 14 d (P21)

rhIL-1ra

Route: Intracerebral administration in the ipsilateral hemisphere

Dose: 3.3 or 5 μg/rat pup

Treatment protocol:

Prophylactic, or in the acute phase post-HI

Administration either before (3.3 μg/rat pup) or immediately after HI (5 μg/rat pup)

In clinical use for other conditions: see above

Degree of neuroprotection

14 d post-HI, assessment of right and left hemisphere dry weights disparities:

- HI duration of 100 min and IL-1ra pretreatment: ~24% neuroprotection (**)

- HI duration of 70 min and IL-1ra post-treatment: no change in mean injury

Cerebral inflammation post-treatment

Not examined

Conclusion

rhIL-1ra mediated neuroprotection, despite being moderate, suggests the pathophysiological significance of IL-1 in neonatal HI.

Liu X et al., 1996

PMID: 8947953

Sprague-Dawley rat

Right CCA ligation

150–195 min 8% O₂

Sacrifice time-point:

5 d (P12)

Platelet-activating factor (PAF) antagonist BN52021

Route: intraperitoneal (i.p.) injection

3 treatment protocols:

Prophylactic, or in the acute phase post-HI

- Pretreatment: 2 serial injections (25 mg/kg/injection), 1 immediately before the onset of hypoxia, and a second one 1 h after the end of hypoxia

- Post-treatment: 2 serial injections (25 mg/kg/injection), 1 immediately after the end of hypoxia, and then 2 h later

- Post-treatment-dose effect: 2 injections, immediately and 2 h after the end of hypoxia- different doses tested (12.5, 25 or 50 mg/kg/injection)

In clinical use for other conditions: PAF antagonists are a heterogenous group of medication, some of which are used for different diseases

Degree of neuroprotection

5 d post-HI, assessed on cresyl violet stained brain slices:

- Incidence of infarction

90% in vehicle-treated rats vs 33% in BN52021-pretreated rats (**) and 30% in BN52021-post-treated rats (*).

- Neuroprotection per region

• Pretreatment: 82% in cortex, 74% in striatum, 90% in hippocampus.

• Post-treatment: 72% in cortex, 46% in striatum, 73% in hippocampus.

5 d post-HI, assessed by interhemispheric weight differences for the post-treatment dose-effect study:

18.9% (NS), 44.8% (*) and 32.5% (*) neuroprotection observed in rats treated with 12.5, 25 or 50 mg/kg/dose compared with controls; differences in neuroprotection among the three doses not statistically different.

Cerebral inflammation post-treatment

Not examined

Conclusion

PAF appears to be one of the acute inflammatory factor mediating perinatal HI brain injury; PAF antagonists may represent a pharmacologic approach to attenuate the severity of HI-brain injury.

Hudome S et al., 1997

PMID: 9128280

Wistar rat

Right CCA ligation

135 min 8% O₂

Sacrifice time-points:

0, 5, and 30 min, and 2, 4, 8, 18, and 42 h

Anti-rat neutrophil serum (ANS) compared to allopurinol

Note: rabbit polyclonal anti-rat neutrophil serum (ANS) is used to deplete neutrophils; comparison to allopurinol, a drug that reduces neutrophil accumulation, and a known neuroprotective agent

Route: i.p. for ANS and s.c. for allopurinol

Dose: Allopurinol 135 mg/kg

Treatment protocol:

In the acute phase post-HI

- 4 treatment groups: allopurinol, neutropenic, both and none

- ANS is injected i.p. at time of surgery

- Allopurinol is injected 15 min post-hypoxia

In clinical use for other conditions: no, ANS is experimental only

Degree of neuroprotection

42 h post-HI, assessed by quantifying brain swelling (wet vs dry weight of posterolateral half of each cerebral hemisphere):

- ~70% (**) reduction in brain swelling by ANS-induced neutropenia and allopurinol. The application of both treatments confers no synergistic effect.

Cerebral inflammation post-treatment

Not examined.

Conclusion by study authors

Neutropenia before the insult is neuroprotective, thus neutrophils may play a role in the pathogenesis of neonatal HI, although the mechanisms remain unclear.

Arvin K et al., 2002 PMID: 12112047

Sprague-Dawley rat

Left CCA ligation

150 min 8% O₂

Sacrifice time-points:

24 h and 1 w

Minocycline

Route: i.p.

Dose: 45 mg/kg

Treatment protocol:

Prophylactic, or in the acute phase post-HI

Single injection immediately before placement in hypoxic chamber, or immediately after removal from hypoxic chamber

In clinical use for other conditions: yes, for various infections

Degree of neuroprotection

7 d post-HI, measurement of the amount of surviving tissue in cresyl violet stained brain slices, and calculation of % tissue loss in HI-IL vs HI-CL hemisphere:

- Minocycline 45 mg/kg before hypoxia: 72% (****), 100% (***) and 85% (****) protection in striatum, hippocampus, and cortex.

- Minocycline 22.5 mg/kg before hypoxia: 61% (NS), 72% (NS) and 71% (*) protection in striatum, hippocampus, and cortex.

- Minocycline 45 mg/kg immediately after hypoxia: similar results as when given before hypoxia.

- Minocycline 45 mg/kg 3 h after hypoxia: 28% (NS), 9% (NS) and 22% (NS) protection in striatum, hippocampus, and cortex.

Cerebral inflammation post-treatment

Not examined

Conclusion

Minocycline at 45 mg/kg provides near complete neuroprotection after neonatal HI only if given immediately before or after HI. Administration 3 h post-HI is not neuroprotective.

Palmer C et al., 2004

PMID: 14739365

Wistar rat

Right CCA ligation

135 min 8% O₂

Sacrifice time-points:

- 42 h post-HI for brain swelling

- 14 d post-HI for brain atrophy

- 0 and 6 h post-HI for analysis of brain energy metabolites

Anti-rat neutrophil serum (ANS)

Route: s.c.

Treatment protocol:

Prophylactic, or in the acute phase post-HI

- before HI group: immediately after carotid ligation

- after HI group: 4–8 h after hypoxia

- 2 corresponding control groups (HI animals exposed to normal rabbit serum)

In clinical use for other conditions: no, experimental only

Degree of neuroprotection

42 h post-HI, measurement of brain swelling:

- In the before HI group, neutropenia reduces brain swelling by 75% (***).

- In the after HI group, no reduction in brain swelling.

- 14 d post-HI, measurement of brain atrophy:

- In the before HI group, neutropenia reduces IL hemisphere atrophy by 61% (*).

- In the after HI group, neutropenia is not protective.

Cerebral inflammation post-treatment

Not examined

Conclusion

Neutrophils play a role in early HI-induced neuropathogenesis. Anti-neutrophil strategies should be applied very early after HI to be neuroprotective.

Jantzie L et al., 2005

PMID: 15647741

Sprague-Dawley rat

Right CCA ligation

150 min 8% O₂

Sacrifice time-points:

- 1 w (P14) for brain stainings

- 1, 2, and 4 h for immunoblotting

Doxycycline (DOXY)

Route: i.p.

Dose: 10 mg/kg

Treatment protocol:

Prophylactic, or in the acute phase post-HI

Injection of DOXY as a one-time dose

- Immediately before HI

- 1 h after HI

- 2 h after HI

- 3 h after HI

In clinical use for other conditions: yes, for various infections

Can cross BBB

Degree of neuroprotection

7 d post-HI, assessment of NeuN immunoreactivity:

In CA1 hippocampus, the average cell loss is ~30% in control animals, whereas it is ~10% in animals treated with DOXY, regardless of injection time-points, which means a ~ 70% neuroprotection.

Cerebral inflammation post-treatment

At 7 d post-HI, trend towards a reduction in the number of ED-1⁺ cells in the IL hippocampus, thalamus and cortex, with a greater extent when DOXY is administered before HI.

Conclusion

DOXY protects the neonatal brain from HI when applied in a short time window post-HI.

Sun Y et al., 2005 PMID: 16040592

Sprague-Dawley rat

Right CCA ligation

150 min 8% O₂

Sacrifice time-points:

3, 7, 14, and 21 d

recombinant human Erythropoietin (rh-EPO)^§

Route: i.p.

Dose: 5 U/g body weight

Treatment protocol:

In the acute and subacute phase post-HI

3 injections, namely injection at 24 h post-HI and for 2 additional days

In clinical use for other conditions: yes, and in clinical trials for infants with CP (NCT# 02811263)

Can cross a dysfunctional blood-brain barrier

Degree of neuroprotection

7, 14 and 21 d post-HI, assessed by measurement of IL and CL wet weight disparities:

~47–77% (***) protection at these 3 time-points.

Cerebral inflammation post-treatment

- rh-EPO abolishes HI-induced rise in IL-1β protein levels in the ipsilateral hemisphere 3, 7, and 14 d post-injury, but does not affect mRNA levels of TNF-α between 3 and 14 d post-HI.

- It limits HI-induced leukocyte infiltration in the parieto-occipital cortex, observed with CD4 and CD68 IHC stainings.

Conclusion

3 doses of rh-EPO is neuroprotective after HI. A potential mechanism of neuroprotection may be through reducing HI-associated elevation in IL-1β and through limiting leukocyte infiltration.

Nijboer C et al., 2008

PMID: 18420952

Wistar rat

Right CCA ligation

120 min 8% O₂

Sacrifice time-points:

1 h, 3 h, 6 h, 12 h, 48 h and 6 w

TAT-NBD, IKK/NF-κB inhibitor

- TAT-NBD: peptide inhibitor of the IKK complex, the NEMO Binding Domain (NBD)-peptide coupled to the protein transduction sequence of HIV-TAT to facilitate cerebral uptake

- mutant TAT-NBD

- biotinylated TAT-NBD

Route: i.p.

Dose: 20 mg/kg

Treatment protocol:

In the acute phase post-HI

administration at various time points post-HI: 0, 3, 6, 9 and 12 h after HI

In clinical use for other conditions: no specific NF-κB inhibitor in use yet, but unrelated drugs may target this signaling pathway

Can cross BBB

Degree of neuroprotection

48 h and 6 w post-HI, assessed by MAP2 staining and hematoxylin-eosin respectively:

- ~40–56% (**) protection at 48 h post-HI if TAT-NBD administered 0/3 h after HI, ~35% (*) if administered 6 h after HI.

- ~75% (***) protection at 6 w post-HI if administered 0/3 h after HI.

- Administration of TAT-NBD at 9 or 12 h post-HI is not neuroprotective.

Cerebral inflammation post-treatment

TAT-NBD treatment immediately after HI does not induce any changes in the HI-induced increase in mRNA levels of cytokine (TNF-α, IL-1β, IL-4, IL-10 and IL-1RA) at 3 h post-HI, even though NF-κB activity is fully inhibited at this time point.

Conclusion

Inhibition of the cerebral IKK/NF-kB pathway early after HI is neuroprotective, but does not involve abrogation of early cytokine expression. This early cytokine induction by neonatal HI may contribute to neuroprotection.

Jin Y et al., 2009

PMID: 19520991

Wistar rat

Right CCA ligation

75 min 8% O₂

Sacrifice time-points:

0, 1 h, 2 h, 4 h, 24 h, 48 h and 4 w

cromolyn, mast cell stabilizer

Route: s.c.

Dose: 50 mg/kg body weight

Treatment protocol:

In the acute phase post-HI

3 injections, i.e. immediately after HI, and 1 and 24 h post-HI

In clinical use for other conditions: yes, for asthma

Degree of neuroprotection

1, 2 and 4 w post-HI, scoring of Fluoro-Jade B stainings, and measurement of IL/CL hemisphere ratio on H&E stained brain slices:

- 61-75% (*) improvement in score of brain damage at the time-points examined.

- Cavitation is absent in all cromolyn treated rats, whereas it is present in 30% and 50% of vehicle-treated animals at 2 and 4 w post-HI, respectively.

- IL/CL ratio restored to normal in cromolyn treated rats (*).

Cerebral inflammation post-treatment

Astrocytic and microglial activation, scored with GFAP and OX-42 antibodies respectively, are significantly reduced at the time points examined.

Conclusion

Mast cells appear to contribute to the inflammatory response to neonatal HI and could be the initiators of the immune response.

Nijboer C et al., 2009

PMID: 19628795

Wistar rat

Right CCA ligation

120 min 8% O₂

Sacrifice time-points:

3, 6, 24 and 48 h

TAT-NBD, IKK/NF-κB inhibitor; TAT-JBD, JNK inhibitor; and etanercept, TNF-α inhibitor

TAT-NBD, mutant TAT-NBD or TAT-JBD

Route: i.p.

Dose: 20 mg/kg for TAT-NBD peptides and 10 mg/kg for TAT-JBD

Treatment protocol: administered 0 and 3 h after HI

Etanercept (a fusion protein of TNF-R2 and human IgG₁):

Route: i.p.

Dose: 5 mg/kg

Treatment protocol:

In the acute phase post-HI

administered directly after HI alone or with TAT-NBD

In clinical use for other conditions: etanercept is used for treating some autoimmune diseases (e.g. rheumatoid arthritis); the other compounds are experimental only

Degree of neuroprotection

48 h post-HI, measurement of CL and IL areas on MAP2 stained brain slices:

- NBD: 75–88% (***).

-JBD: 25% (*).

- NBD + JBD: 50% (***).

- Etanercept: 25% (*).

- NBD + Etanercept: 44% (* vs NBD).

Cerebral inflammation post-treatment

- Combined NF-κB inhibition and JNK/AP-1 prevents HI-induced TNF-α production and reduces neuroprotection.

- Etanercept alone has a significant neuroprotective effect, but not as great as that provided by NF-κB inhibition.

- Etanercept treatment reduces the protective effect of TAT-NBD.

Conclusion

JNK/AP-1 inhibition and TNF-α inhibition reduce the neuroprotective effect of NF-κB inhibition: after HI is mediated in part by JNK/AP-1 activity and TNF-α production.

Fathali N et al., 2010

PMID: 20029340

Sprague-Dawley rat

P10

Right CCA ligation

120 min 8% O₂

Sacrifice time-points:

72 h, 2 w, or 6 w

NS398, selective cyclooxygenase-2 (COX-2) inhibitor

Route: i.p.

Dose: 2 dosage regimens, either 10 mg/kg (NS-10 group), or 30 mg/kg (NS-30 group)

Treatment protocol:

In the acute and subacute phase post-HI

six injections (1, 6, 24, 36, 48, and 60 h) after hypoxia

In clinical use for other conditions: going out of clinic due to adverse cardiac events

Degree of neuroprotection

2 and 6 w post-HI, measurement of IL/CL weight ratio:

- NS-10 group: 63% at 2 w (*) and 57% (*) at 6 w.

- NS-30 group: 79% at 2 w (*) and 75% (*) at 6 w.

6 w post-HI, behavior analysis:

improvement in, or restoration of many HI-induced neurobehavioral deficits in NS-10 and NS-30 treated animals.

Cerebral inflammation post-treatment

- COX-2 immunopositive signals (western blotting and IHC staining of ipsilateral brain) are significantly reduced in NS398 treated HI animals in comparison to HI-vehicle animals.

- At 72 h post-HI, IL-6 staining and concentration in ipsilateral brain is increased in HI-vehicle exposed rats, and significantly reduced by NS-30. Qualitative evaluation of single immunofluorescent staining for Iba1, CD68 and MPO in ipsilateral brain indicates that NS30 attenuates significantly HI-induced increase in these markers.

Conclusion

COX-2 inhibition is neuroprotective; effects may be mediated by anti-inflammatory actions, through IL-6 reduction, decreased activation of microglia and of infiltration of macrophages and neutrophils.

Cikla U, 2016

PMID: 26857490

C57BL/6 J mice

- P9 “P9 neonatal mice/brain”

- P30 “P30 juvenile mice/brain”

Left CCA ligation

50 min 10% O₂

Sacrifice time-points:

2, 9 and 60 d

Minocycline

Route: i.p.

Dose: 40 mg/kg

Treatment protocol:

In the acute phase post-HI

Two injections, one at 2 h and the second one at 24 h post-HI

In clinical use for other conditions: see above

Degree of neuroprotection

2 and 9 d post-HI, scoring of damage on MAP2 stained brain slices:

- P9 neonatal brain: 73% (*) and 87% (*) protection at 2 and 9 d post-HI.

- P30 juvenile brain: no protection at both days; of note, HI-induced brain damage less severe at P30 than at P9.

Brain atrophy at 9 and 60 d post-HI by T2-weighted MRI:

- P9 neonatal brain: no significant impact on volume loss at both days.

- P30 juvenile brain: no significant impact on volume loss at 9 d, but a slight impact at 60 d.

Hippocampal learning by Morris water maze test:

- P9 neonatal brain: no amelioration in HI-induced learning deficits.

- P30 juvenile brain: significant improvement.

Cerebral inflammation post-treatment

Impact of minocycline on total counts of microglia (CD11b⁺/CD45⁺)

- Significantly suppresses HI-induced increase in microglia counts in the IL hippocampus from P9 and P30 brains 2 and 9 d post-HI; this is also observed in the cortex and striatum from P9 brains, but less in the same regions from P30 brains.

Impact of minocycline on percentage of activated microglia (CD45^+med/total microglia)

- 2 d post-HI, in the IL hippocampus, induces ~70% reduction in the proportion of activated microglia in both neonatal and juvenile brains.

- 9 d post-HI in the IL cortex and striatum

- P9 neonatal brain: significant decrease in the percentage of activated microglia

- P30 juvenile brain: no impact of minocycline on HI-induced late microglia activation

Conclusion

Minocycline has different neurological outcome depending on the age at which HI-injury is induced. Early improvements in neurologic injury do not necessarily predict long-term improvements in neurologic function.

Herz J et al., 2018

PMID: 30127782

C57BL/6 J mice

Right CCA ligation

60 min 10% O₂

Sacrifice time-points:

1 w (P16)

FTY720 (Fingolimod), a sphingosin-1-phosphate analog that reduces peripheral lymphocytes

Route: i.p.

Dose: 1 mg/kg body weight

Treatment protocol:

In the acute phase post-HI

single injection within 20 min after hypoxia

In clinical use for other conditions: yes, for multiple sclerosis

Degree of neuroprotection

1 w post-HI, scoring of injury in cresyl violet stained brain slices:

- Significant worsening of neuropathology in FTY720-treated animals versus saline-exposed mice.

Cerebral inflammation post-treatment

- FTY720 treatment induces a slight but not significant increase in Iba1 protein levels (assessed by western blot).

- Despite FTY720 induced depletion of circulating T cells, the total cerebral leukocyte infiltration assessed qualitatively by IHC is unaffected by FTY720. This is explained by an increased infiltration of innate immune cells, mainly neutrophils and inflammatory macrophages.

Conclusion

Pharmacological mediated T cell depletion increases HI-induced brain injury; this contrasts with the neuroprotective effect of FTY720 observed in adult models of stroke. Thus, neonatal T cells may promote endogenous neuroprotection.

All sacrifice time-points are given in post-HI

H&E: hematoxylin-eosin

BBB: Blood brain barrier

^† Percent neuroprotection was either reported by study authors, either calculated by reviewers using the formula 100*(1-%damage in treated animals/%damage in vehicle treated animals), as in [146]. The significance is that reported by authors, and unless indicated, represents the significance of HI drug-treated animals versus HI vehicle-treated animals. Significance values * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001

^§ Various rhEPO treatment protocols have been tested in the P7 rodent model of neonatal HI (for a review, see [143]). This study is included in the table because it is the only one that reports on CD68 immunostaining in the ipsilateral hemisphere from HI-exposed animals. The study authors attribute CD68 staining to infiltrating leukocytes, but it could also be microglia/macrophages