(Stroke. 1999;30:1916-1924.)
© 1999 American Heart Association, Inc.
Original Contributions |
Inflammatory Responses in the Cerebral Cortex After Ischemia in the P7 Neonatal Rat
From the Université René Descartes, Paris, France.
Correspondence to C. Charriaut-Marlangue, INSERM U29, 123 bd de Port-Royal, 75014 Paris, France. E-mail cm{at}u29.cochin.inserm.fr
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferencesIntroduction References |
|---|
Background and Purpose—The contribution of inflammatory response to the pathogenesis of ischemic lesions in the neonate is still uncertain. This study described the chronological sequence of inflammatory changes that follow cerebral ischemia with reperfusion in the neonatal P7 rat.
Methods—P7 rats underwent left middle cerebral artery electrocoagulation associated with 1-hour left common carotid artery occlusion. The spatiotemporal pattern of cellular responses was characterized immunocytochemically with the use of antibodies against rat endogenous immunoglobulins to visualize the area of the breakdown of the blood-brain barrier. Infiltration of neutrophils and T lymphocytes was demonstrated by antibodies against myeloperoxidase and a pan-T cell marker, respectively. Antibodies ED1 and OX-42 were applied to identify microglial cells and macrophages. The response of astrocytes was shown with antibodies against glial fibrillary acidic protein. Cell survival was assessed by Bcl-2 expression. Cell death was demonstrated by DNA fragmentation with the use of the terminal deoxynucleotidyl transferase–mediated dUTP biotin nick end labeling (TUNEL) assay and Bax immunodetection.
Results—Endogenous immunoglobulin extravasation through the blood-brain barrier occurred at 2 hours of recirculation and persisted until 1 month after ischemia. Neutrophil infiltration began at 24 hours and peaked at 72 to 96 hours (30±3.4 neutrophils per 0.3 mm2; P<0.0001), then disappeared at 14 days after ischemia. T cells were observed between 24 and 96 hours of reperfusion. Resident microglia-macrophages exhibited morphological remnants and expressed the cell death inhibitor Bcl-2 at 24 hours of recirculation. They became numerous within the next 48 hours and peaked at 7 days after ischemia. Phenotypic changes of resident astrocytes were apparent at 24 hours, and they proliferated between 48 hours and 7 days after ischemia. Progressively inflammatory cells showed DNA fragmentation and the cell death activator Bax expression. Cell elimination continued until there was a complete disappearance of the frontoparietal cortex.
Conclusions—These data demonstrate that perinatal ischemia with reperfusion triggers acute inflammatory responses with granulocytic cell infiltration, which may be involved in accelerating the destructive processes.
Key Words: brain injuries • ischemia • leukocytes • newborn • reperfusion • rats
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesIntroduction References |
|---|
Perinatal ischemic brain injury may lead to the death of neonates and important neurological impairment manifested as cerebral palsy and mental retardation in the surviving infants.1 2 3 This is due to the fact that the basal ganglia as well as certain regions of neocortex are relatively vulnerable to injury in the neonatal period.4 Perinatal asphyxia, with its attendant bradycardia and hypotension, is an ischemic condition that is followed by reperfusion on resuscitation.
Reperfusion after an ischemic episode elicits in adult brains the appearance of deleterious blood leukocytes, potentially a source of toxic free radicals and cytokines causing inflammatory reactions and leading to a delayed death of neurons and the expansion of cerebral damage (for reviews, see References 5 and 65 6 ). In contrast, the physiological events that follow reperfusion in the neonate have not been largely examined. With the use of the classic model of Rice et al7 in 7-day-old rats, neuronal death was found to be associated with an activation of both microglia and astrocytes, while granulocytic cells were not involved.8 9 10 It is presently unclear whether those effects result from the combination of both processes (ischemia and hypoxia) or from ischemic insult alone. To investigate the mechanisms of reperfusion injury, we recently developed a model of stroke by transient unilateral focal ischemia in P7 rats by permanent left middle cerebral artery (MCA) occlusion with 1-hour occlusion of the left common carotid artery. Ischemia with reperfusion in the anastomoses via the carotid artery produced a well-delineated cortical infarct at 48 hours of reperfusion11 and neuronal death with features characteristic of apoptosis.11 12
This study was designed to investigate inflammatory responses in this model between 2 hours and 3 months of reperfusion. Our data strongly suggest that inflammatory cells may be involved in promoting secondary cell death in neonatal ischemia with reperfusion.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesIntroduction References |
|---|
Animals and Neonatal Ischemia
Experiments involving animals were approved by the French Ethical Science Committee (statement No. 04223). Experiments were performed in 7-day-old Wistar rats (weight, 15 to 23 g) of both sexes, as previously described.11 Rat pups were anesthetized with an injection of chloral hydrate (300 mg/kg IP). After 15 minutes, rats were placed on their back, and a median incision was made in the neck to expose the left common carotid artery. Rats were then placed on the right side, and an oblique skin incision was made between the ear and the eye. After excision of the temporal muscle, the cranial bone was removed from the frontal suture to a level below the zygomatic arch. Then the left MCA, exposed just after its apparition over the rhinal fissure, was coagulated at the inferior cerebral vein level. After this procedure, a clip was placed to occlude the left common carotid artery and was removed after 1 hour. Carotid blood flow restoration was verified with the aid of a microscope. Both neck and cranial skin incisions were then closed. The surgical procedure lasted approximately 20 minutes. During surgery, body temperature was maintained at 37°C to 38°C. Rat pups were placed in an incubator maintained at 37°C until they woke up, and then they were transferred to their mother for long-time survival.
Tissue Preparation
Each of the following survival times after reperfusion studied
was represented by a group of 5 animals: 6, 24, 72, and 96
hours; 7 and 14 days; and 1 and 3 months. At each point, animals were
perfused transcardially with warm heparinized saline followed by a
phosphate-buffered solution (0.1 mol/L, pH 7.4) containing 4%
paraformaldehyde. Brains were immediately removed and
postfixed for 1 hour in the same fixative solution at room temperature.
Then they were placed in a phosphate-buffered solution containing 10%
sucrose for 2 days at 4°C. The brains were frozen in isopentane at
-40°C. They were then stored at -80°C until used. For control, 2
sham age-matched rats were killed and processed in the same way.
Coronal cryostat sections (10 µm thick) were collected on
gelatin-coated slides. Cresyl violet was used to identify the site of
the lesion and to evaluate degenerating cells.
Immunocytochemistry
Antisera
A mouse monoclonal antibody against glial fibrillary acidic
protein (GFAP) (1:200, Boehringer Mannheim) was used to
visualize astrocytes. Rat microglia and brain macrophages were
immunostained by the mouse monoclonal antibody OX-42
(1:500, Serotec) and ED1 (1:500, Serotec). OX-42 recognizes the
complement C3bi receptor expressed by monocytes/macrophages and
activated ramified and ameboid microglia. ED1 antibodies
label most rat macrophages, peripheral blood
monocytes, and activated microglia.13 The area of
leakage of the blood-brain barrier (BBB) was identified by
immunodetection of rat endogenous immunoglobulins in brain
parenchyma (1:200, Dako). Rabbit antibodies against myeloperoxidase
(MPO) (1:200, Sigma) were used to visualize polymorphonuclear
leukocytes (PMNL). Mouse monoclonal antibodies against rat T lymphocyte
(Pan-T, 1:200, Serotec) were used to identify infiltrating T
lymphocytes. Bcl-2 antibodies were purchased from Boehringer
Mannheim and Bax (P-19) antibodies from Santa Cruz (Tebu). They
recognized p26 and p21 proteins to identify key regulators of
apoptosis in surviving and dying cells, respectively.
Immunostaining Procedure
Sections were incubated overnight at 4°C in the first antibody
at the dilution indicated above in PBS 0.1 mol/L containing 2% BSA,
0.3% Triton X-100. The secondary (anti-rabbit or anti-mouse IgG)
biotinylated antibodies (1:200 to 1:400 dilution, Dako) were visualized
by the avidin-biotin peroxidase (Elite ABC kit, Vectastain Vector,
Byosis). The peroxidase activity was evidenced with the use of 0.1
mol/L phosphate buffer containing 0.025% 3,3'-diaminobenzidine (DAB)
and 0.02% hydrogen peroxide. Nonspecific peroxidase activity was
abrogated by incubating the sections in 2% hydrogen peroxide in 10%
methanol at the appropriate stage. Some immunostained
sections were counterstained with hematoxylin for localization of the
labeled cells. In double-labeling experiments, sections were first
incubated with antibodies against GFAP, OX-42, or ED1 and revealed by
the avidin-biotin alkaline phosphatase (ABC kit, Vectastain Vector)
with the Substrate Kit III (Vector Blue). They
were then incubated with anti-Bax and visualized with peroxidase and
DAB.
In Situ Labeling of Fragmented DNA
Coronal cryostat sections were processed for terminal
deoxynucleotidyl transferase–mediated dUTP biotin
nick end labeling (TUNEL) assay, as previously reported.14
Briefly, sections were incubated with terminal
deoxynucleotidyl transferase (0.2 U/µL; Gibco)
and biotin-16-dUTP (20 µmol/L; Boehringer Mannheim),
then visualized with streptavidin-biotin-peroxidase complex and
diaminobenzidine.
Quantification of Neutrophils
Quantification of neutrophils was performed with the use of a
x40 objective and a camera lucida attached to the microscope. At
different times after ischemia, in the 5 ischemic
animals, intraparenchymal MPO-positive neutrophils were counted in a
0.3-mm2 circular area on 3 sections containing
the infarct. The statistical significance of the results was evaluated
by the unmatched Student's t test.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesIntroduction References |
|---|
Sections used in the figures are representative of the 5 animals analyzed in each group at each time period.
Histopathology
Figure 1
shows the cytoarchitecture
and cell morphology of the cortex from control (Figure 1A) and
ischemic (Figure 1B through 1D) rat pup brains after
examination of cresyl violet–stained sections. At 72 hours of
reperfusion (Figure 1B), the infarcted area collapsed and
demonstrated cells with coarse chromatin clumping and nuclear pyknosis
(black arrow in Figure 1E). At the periphery of the infarct, a
more marked hypercellular band (white curved arrows in Figure 1B) appeared and showed several mitotic figures (white curved
arrow in Figure 1F). One week after the ischemic insult,
the infarcted area cleared progressively of neuronal perikarya.
Cortical upper layers disappeared, and a cavity was formed (Figure 1C, black arrow). By 2 weeks of reperfusion, the ipsilateral
hemisphere appeared reduced compared with the contralateral hemisphere
(not shown). The loss of the frontoparietal cortex was completed 3
months after recovery (Figure 1D).
|
Early BBB Impairment, PMNL, and Lymphocyte Infiltration
Endogenous immunoglobulin extravasation was only
observed in the ischemic ipsilateral hemisphere and appeared as
early as 2 hours after reperfusion in the core of the infarct. At 72
hours, the area of BBB leakage was larger than the infarcted area
(Figure 2A). Altered cells within
the infarct (Figure 2B), glial cells at the periphery of the
infarct (Figure 2C), and nonaffected neurons located in regions
without the BBB and occasionally in the substantia nigra (Figure 2D) were immunostained by anti-rat immunoglobulins.
Anti-rat immunoglobulin immunostaining was still
observed 1 month after reperfusion in the ipsilateral hemisphere, but
not thereafter.
|
PMNL were detected in the ischemic tissue at 24 hours of
reperfusion. At this point, they were essentially observed in arachnoid
spaces and associated with intraparenchymal blood vessels within and
near the lesioned area (not shown). Progressively, PMNL invaded the
upper layers of infarcted cortex from 48 to 96 hours of recovery
(Figure 2E
and 2F). Intraparenchymal neutrophils peaked at 72 to
96 hours of reperfusion (Figure 3). No
PMNL were observed after 2 weeks of reperfusion. Infiltrating
lymphocytes (pan-T immunoreactive cells) were transiently observed
between 24 and 96 hours of recovery and were preferentially located in
the white matter of the ipsilateral hemisphere, notably the corpus
callosum (Figure 2G), the internal capsule, and often in close
vicinity to blood vessels.
|
Time Course of Microglia/Macrophage Responses
Activated OX-42–immunostained
microglia/macrophages in neonatal ischemic rat brain
were detected at the periphery of the infarcted region at 72 hours of
reperfusion (Figure 4A). They
increased in number and progressively invaded the injured tissue
between 96 hours and 1 week of reperfusion (Figure 4B) and
expressed the survival promoter Bcl-2.15
Bcl-2–immunoreactive cells showed cell bodies and numerous long, thin
processes (called ramified microglia) (Figure 4C and 4D) or
stout processes (called ameboid microglia) (Figure 4E).
Quiescent microglia in sham pups was not immunostained by
Bcl-2 antibodies (not shown). OX-42–immunostained
microglia persisted around the cavity several weeks after
ischemia.
|
ED1 antibody identified macrophages, round cells that had a
smooth surface and lacked processes; they appeared first in the lateral
ventricle and migrated along the corpus callosum at 24 hours of
recovery (Figure 4F). A prominent increase in their
accumulation was observed between 72 hours and 1 week of reperfusion,
and these cells invaded the infarcted area (Figure 4G).
Pseudopodic and ramified ED1-positive microglia/macrophages
remained present in the infarcted area at later times of
reperfusion.
Time Course of Astroglial Cell Responses
During the first month of life, control rat brains showed
GFAP-positive astrocytes predominantly in the corpus callosum and the
internal capsule. The molecular layer of the cortex and perivascular
spaces showed weak immunostained GFAP-positive astrocytes
(Figure 5A through 5C). The
infarcted cortical area of ischemic rats showed increased GFAP
immunoreactivity 24 to 48 hours after reperfusion. Reactive astrocytes
formed a dense network delineating the infarcted area (Figure 5D and 5E). Reactive GFAP-immunostained cells have a small
soma and long processes directed toward the subpial spaces and the core
of the infarct (Figure 5F). One week after the ischemic
insult, strongly GFAP-immunostained astrocytes were
observed at the periphery and within the infarct (Figure 5G).
After the second week of recovery, reactive astrocytes within the
infarct showed features of degradation, including a decrease in GFAP
immunostaining in fragmented processes and residual
star-shaped cell elements (Figure 5H). From 14 days until 2
months after reperfusion, astrocyte processes closely surrounded the
smooth-walled cavity (Figure 5I).
|
Cell Death: Did Glial Cells Undergo Apoptosis?
Cells exhibiting DNA fragmentation were shown as early as 6 hours;
they increased and peaked at 24 to 96 hours.11 During the
next 2 weeks, the infarct developed a smooth-walled cavity (Figure 1), and TUNEL-positive cells were observed around this cavity
(Figure 6A). These cells, with
different morphology, exhibited the death promoter gene Bax, expressed
by dying cells,16 as demonstrated by
immunostaining of adjacent sections (Figure 6B).
At 1 month of recirculation, Bax-positive cells were detected at the
periphery of the infarct (Figure 6C), but these cells displayed
a glial morphology (Figure 6D). The staining of sections with
both Bax and GFAP or OX-42 antibodies revealed the presence of
double-labeled astrocytes (Figure 7A) and
microglia (Figure 7B) at the periphery of the infarct between 14
and 30 days of recovery.
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesIntroduction References |
|---|
The present study shows that ischemia/reperfusion in immature rats induces progressive tissue destruction, leading to a well-delineated lesion in the ipsilateral cortex at 3 months after recovery. BBB impairment followed by gliosis and infiltration of leukocytes provides evidence of a strong local inflammatory response (summarized in the Table
). At later times
of reperfusion, cells integrated in the inflammatory response were
eliminated through an apoptotic pathway.
|
The BBB was reported to contribute to the progression of the cellular injury, as suggested in other models of cerebral ischemia (for review, see Reference 1515 ). The labeling of neurons by anti-rat immunoglobulins within the ipsilateral hemisphere may reflect a nonspecific uptake of extravasating immunoglobulins rather than specific recognition, as previously described in other pathological processes16 ; that of microglial cells may be due to the overexpression of the Fc receptor during their activation.17 BBB disruption may also induce an entry of toxic molecules, such as albumin and excitatory amino acids. There has been increasing recognition that the cerebrovascular endothelium participates in immune responses as a target for cytokine action and is a source of arachidonic acid metabolites,18 free radicals, and nitric oxide.19 Furthermore, endothelial cells participate in immune responses by the regulation of intercellular adhesion molecule 1,20 which plays a key role in leukocyte recruitment and subsequent brain injury.21
The first immune cells to appear in the ischemic immature tissue are T lymphocytes and PMNL. Abundant22 and small23 T-cell infiltrates have been noted after MCA occlusion in the adult rat brain. PMNL were observed to be accumulated in the subarachnoid spaces and blood vessels 24 hours after reperfusion and progressively invaded the different layers of the infarcted cortex. Perivascular intraparenchymal neutrophil migration into the ischemic brain has been reported to begin as early as 624 and 2425 hours after recirculation following ischemia/reperfusion in adult rat. In contrast, minimal neutrophilic response9 and lack of neutrophil extravasation during the evolution of the infarct26 were reported following neonatal hypoxia/ischemia. Nevertheless, neutrophil depletion in such a model reduced brain swelling at 42 hours of recovery by approximately 70%, which was comparable to the protective effect of allopurinol.26 PMNL are known to be integrated into the acute inflammatory response and possess extensive cytotoxic capacities, supporting their pathological role in destructive processes. They represent an important biological source of oxygen-derived free radicals and generate nitric oxide underlying oxidant-mediated tissue injury.27 28 We recently reported that perivascular reactions mediated by nitric oxide and peroxynitrites in T lymphocytes and polymorphonuclear cells are important in the cascade of events that lead to brain oxidative stress in neonatal ischemia.29 In addition, NG-nitro-L-arginine methyl ester, an inhibitor of nitric oxide synthase, reduced infarct volume in a filament model of transient MCA occlusion in the rat pup.30
Our results demonstrated a microglial activation elicited by
ischemia in immature rats that was first detected at 3 days
after injury. The presence of ED1-positive
microglia-macrophages in the ipsilateral
periventricular area has been previously reported after
hypoxia/ischemia in P7 rats.31 A rapid,
relatively widespread microglial response was also demonstrated after
hypoxia/ischemia by the use of major histocompatibility
complex II (OX-6) and complex I (OX-18) antibodies,10 32
whereas macrophage infiltration may be delayed or
absent.32 A relationship between delayed neuronal loss in
tissues neighboring sites of ischemia and cytotoxin secretion
has been suggested.33 Activated microglia may
release a variety of cytokines, including interleukin-1ß and
tumor necrosis factor-
, known to activate astrocyte
proliferation,34 35 leukocyte
infiltration,36 37 and brain edema.38
However, their role in the progression of ischemic lesion
remains uncertain.39 The association of activated
microglia with nuclear ghosts in the infarcted area also suggests that
they contribute to the removal of cell death–associated debris induced
by ischemic injury. Activated microglia after
ischemia may act as mediators of further injury and/or as
cleansing scavengers and repair agents.40 Microglial cells
seem to express the death inhibitor protein Bcl-2.
Furthermore, the delayed expression of the cell death promotor Bax from
2 weeks until 1 month after injury suggests their death by
apoptotic features, as demonstrated in human atherosclerotic
lesions.41 Therefore, the balance between
proapoptotic (Bax proteins) and antiapoptotic (Bcl-2)
elements may also account for death or survival of glial cells,
respectively. Evidence of interleukin-1ß–converting
enzyme–like immunoreactivity in microglial cells was recently
reported, which suggests a potential role for caspases in
ischemic damage through mediation of an inflammatory
response.42 Apoptosis, through its several steps,
thus appears to be one mechanism by which activated microglia
are gradually eliminated after cerebral ischemia and by which
steady state of microglial cell numbers is achieved in vivo.
Reactive astrocytes are believed to reestablish a protective barrier, the glial-limiting membrane.43 Astrocytic changes in the present model of neonatal stroke could be directed to newly formed glia limitans around the cortical cavity. The temporal pattern of astrocyte responses indicates that reactive astrocytes may assist ischemic brain repair by stabilizing the tissue surrounding neuronal injury.44 Early morphological remnants of resident astrocytes may reflect changes in ionic concentrations and excitatory amino acids in the area of neuronal depletion. It is presently unclear whether these astrocytes migrate into injured areas or locally proliferate. However, our previous data suggested that glial cells at the periphery of the infarct expressed proliferating cell nuclear antigen,12 known to be involved in protective and proliferative processes. At later times of recirculation, astrocytes exhibited DNA fragmentation and expressed the cell death promotor Bax, suggesting that their downregulation occurred through apoptosis, compared with microglial cells.
In conclusion, our results show that ischemia without hypoxia in immature animals triggers microglial cell activation, astrocytic response, and granulocytic infiltration, similar to those seen in adult brain (for comparison, see References 45 through 4845 46 47 48 ). The main discrepancy between our results and that found after neonatal hypoxia/ischemia was neutrophil involvement. Our results, however, did not establish a cause-and-effect relationship between the inflammatory markers and the progression of histological damage. To investigate such functional outcome, experiments with inflammatory mediator antagonists (free radical scavengers, anti–adhesion molecule antibodies, cytokine antagonists)49 would be of interest.
Received February 4, 1999; revision received April 29, 1999; accepted June 2, 1999.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesIntroduction References |
|---|
1. Younkin D. Hypoxic-ischemic brain injury of the newborn: statement of the problem and overview. Brain Pathol. 1992;2:209–210.[Medline] [Order article via Infotrieve]
2. Gray P, Tudehope T, Masel J, Burns Y, Mohay H, O'Callaghan M, Williams G. Perinatal hypoxic-ischaemic brain injury: prediction of outcome. Dev Med Child Neurol. 1993;35:965–973.[Medline] [Order article via Infotrieve]
3. Volpe J. Neurology of the newborn. In: Neurology of the Newborn. Philadelphia, Pa: WB Saunders; 1987.
4. Friede R. Perinatal lesions of gray matter. In: Friede RL, ed. Developmental Neuropathology. Göttingen, Germany: Springer-Verlag; 1989:82–97.
5. Feuerstein G, Liu T, Barone F. Cytokines, inflammation and brain injury: role of tumor necrosis factor. Cerebrovasc Brain Metab Rev. 1994;6:341–360.[Medline] [Order article via Infotrieve]
6. Fellman V, Raivio K. Reperfusion injury as the mechanism of brain damage after perinatal asphyxia. Pediatr Res. 1997;41:599–606.[Medline] [Order article via Infotrieve]
7. Rice J, Vannucci R, Brierley J. The influence of immaturity on hypoxia-ischemic brain damage in the rat. Ann Neurol. 1981;9:131–141.[Medline] [Order article via Infotrieve]
8. Towfighi J, Yager J, Housman C, Vannuci R. Neuropathology of remote hypoxic-ischemic damage in the immature rat. Acta Neuropathol (Berl). 1991;81:578–587.[Medline] [Order article via Infotrieve]
9. Towfighi J, Zec N, Yager J, Housman C, Vannucci R. Temporal evolution of neuropathologic changes in immature rat model of cerebral hypoxia: a light microscopic analysis. Acta Neuropathol (Berl). 1995;90:375–386.[Medline] [Order article via Infotrieve]
10. McRae A, Gilland E, Bona E, Hagberg H. Microglia activation after neonatal hypoxia-ischemia. Dev Brain Res. 1995;84:245–252.[Medline] [Order article via Infotrieve]
11.
Renolleau S, Aggoun-Zouaoui D, Ben-Ari Y,
Charriaut-Marlangue C. A model of transient unilateral focal
ischemia with reperfusion in the P7 neonatal rat: morphological
changes indicative of apoptosis. Stroke. 1998;29:1454–1461.
12. Renolleau S, Benjelloun N, Ben-Ari Y, Charriaut-Marlangue C. Regulation of apoptosis-associated proteins in cell death following transient focal ischemia in rat pup. Apoptosis. 1997;2:368–376.[Medline] [Order article via Infotrieve]
13. Milligan C, Cunningham T, Levitt P. Differential immunochemical markers reveal the normal distribution of brain macrophages and microglia in the developing rat brain. J Comp Neurol. 1991;314:125–135.[Medline] [Order article via Infotrieve]
14. Charriaut-Marlangue C, Margaill I, Represa A, Popovici T, Plotkine M, Ben-Ari Y. Apoptosis and necrosis following reversible focal ischemia: an in situ DNA fragmentation analysis. J Cereb Blood Flow Metab. 1996;16:186–194.[Medline] [Order article via Infotrieve]
15. Betz A, Dietrich W. Blood-brain barrier: dysfunction in cerebral ischemia. In: Ginsberg M, Bogousslavsky J, eds. Cerebrovascular Disease: Pathophysiology, Diagnosis and Management. Philadelphia, Pa: Blackwell Science; 1998:358–370.
16. Rhodes R. Evidence of serum-protein leakage across the blood-brain barrier in the acquired immunodeficiency syndrome. J Neuropathol Exp Neurol. 1991;50:171–183.[Medline] [Order article via Infotrieve]
17. Vedeler C, Ulvestad E, Grundt I, Conti G, Nyland H, Matre R, Pleasure D. Fc receptor of immunoglobulin for IgG (FcR) an rat microglia. J Neuroimmunol. 1994;49:19–24.[Medline] [Order article via Infotrieve]
18. Maurer P, Moskowitz M, Levine L, Melamed E. The synthesis of prostaglandins by bovine cerebral microvessels. Prostagland Med. 1980;4:153–161.
19. Beckman J, Ye Y, Chen J, Conger K. The interactions of nitric oxide with oxygen radicals and scavengers in cerebral ischemic injury. In: Siesjö BK, Wieloch T, eds. Advances in Neurology: Cellular and Molecular Mechanisms of Ischemic Brain Damage. Philadelphia, Pa: Lippincott-Raven; 1996:339–354.
20. Chopp M, Zhang R, Chen H, Li Y, Jiang N, Rusche JR. Postischemic administration of an anti-Mac-1 antibody reduces ischemic cell damage after transient middle cerebral occlusion in rats. Stroke. 1994;25:869–876.[Abstract]
21. Hallenbeck J, Kochanek P. Inflammatory responses in cerebral ischemia: role of leukocytes. In: Ginsberg M, Bogousslavsky J, eds. Cerebrovascular Disease: Pathophysiology, Diagnosis and Management. Philadelphia, Pa: Blackwell Science; 1998:489–506.
22. Schroeter M, Jander S, Witte O, Stoll G. Local immune responses in the rat cerebral cortex after middle cerebral artery occlusion. J Neuroimmunol. 1994;55:195–203.[Medline] [Order article via Infotrieve]
23. Morioka T, Kalehua A, Streit W. Characterization of microglial reaction after middle cerebral artery occlusion in rat brain. J Comp Neurol. 1993;121:123–132.
24. Clark R, Lee E, White R, Jonak Z, Feuerstein G, Barone F. Reperfusion following focal stroke hastens inflammation and resolution of ischemic injured tissue. Brain Res Bull. 1994;35:387–392.[Medline] [Order article via Infotrieve]
25. Garcia J, Liu K, Yoshida Y, Lian J, Chen S, Del Zoppo G. Influx of leukocytes and platelets in an evolving brain infarct. Am J Pathol. 1994;144:188–199.[Abstract]
26. Hudome S, Palmer C, Roberts R, Mauger D, Housman C, Towfighi J. The role of neutrophils in the production of hypoxic-ischemic brain injury in the neonatal rat. Pediatr Res. 1997;41:607–616.[Medline] [Order article via Infotrieve]
27.
Traytsman R, Kirsch J, Koehler R. Oxygen radical
mechanisms of brain injury following ischemia and reperfusion.
J Appl Physiol. 1991;71:1185–1195.
28.
Chan P. Role of oxidants in ischemic brain
damage. Stroke. 1996;27:1124–1129.
29. Coeroli L, Renolleau S, Arnaud S, Plotkine D, Cachin N, Plotkine M, Ben-Ari Y, Charriaut-Marlangue C. Nitric oxide formation and perivascular nitrotyrosine following focal ischemia in immature rats. J Neurochem. 1998;70:2516–2525.[Medline] [Order article via Infotrieve]
30. Ashwal S, Cole D, Osborne S, Osborne T, Pearce W. L-NAME reduces infarct volume in a filament model of transient middle cerebral artery occlusion in the rat pup. Pediatr Res. 1995;38:652–656.[Medline] [Order article via Infotrieve]
31. Ivacko J, Sun R, Silverstein F. Hypoxic-ischemic brain injury induces an acute microglial reaction in perinatal rats. Pediatr Res. 1996;39:39–47.[Medline] [Order article via Infotrieve]
32. Ohno M, Aotani H, Shimada M. Glial responses to hypoxic/ischemic encephalopathy in neonatal rat cerebrum. Dev Brain Res. 1995;84:294–298.[Medline] [Order article via Infotrieve]
33. Giulian D, Vaca K. Inflammatory glia mediate delayed neuronal damage after ischemia in the central nervous system. Stroke. 1993;24(suppl I):I-184–I-190.
34.
Giulian D, Lachman L. Interleukin-1 stimulation of
astroglial proliferation after brain injury. Science. 1985;228:497–499.
35. Giulian D, Woodward J, Young D, Krebs J, Lachman L. Interleukin-1 injected into mammalian brain stimulates astrogliosis and neovascularisation. J Neurosci. 1988;8:2485–2490.[Abstract]
36. Kishimoto T, Rothlein R. Integrins, ICAMs, and selectins: role and regulation of adhesion molecules in neutrophil recruitment to inflammatory sites. Adv Pharmacol. 1994;25:117–169.
37. Montefort S, Holgate S. Adhesion molecule and their role in inflammation. Respir Med. 1991;85:91–99.[Medline] [Order article via Infotrieve]
38. Gordon C, Merchant R, Marmarou A, Rice C, Marsh T, Young H. Effect of murine recombinant interleukin-1 on brain edema in the rat. Acta Neurochir (Wien). 1990;51:268–270.
39. Giulian D. Ameboid microglia as effectors of inflammation in the central nervous system. J Neurosci Res. 1987;18:155–171.[Medline] [Order article via Infotrieve]
40. Giulian D, Chen J, Ingeman J, George J, Noponen M. The role of mononuclear phagocytes in wound healing after traumatic brain injury to adult mammalian brain. J Neurosci. 1989;9:4416–4429.[Abstract]
41. Bjorkerud S, Bjorkerud B. Apoptosis is abundant in human atherosclerotic lesions, especially in inflammatory cells (macrophages and T cells), and may contribute to the accumulation of gruel and plaque instability. Am J Pathol. 1996;149:367–380.[Abstract]
42.
Bhat R, DiRocco R, Marcy V, Flood D, Zhu Y, Dobrzanski
P, Siman R, Scott R, Contreras P, Miller M. Increased expression of
IL-1beta converting enzyme in hippocampus after ischemia:
selective localization in microglia. J Neurosci. 1996;16:4146–4154.
43. Reier P. Gliosis following CNS injury: the anatomy of astrocytic scars and their influences on axonal elongation. In: Fedoroff S, Vernadakis A, eds. Astrocytes, Cell Biology and Pathology of Astrocytes. New York, NY: Academic Press; 1986:263–324.
44. Montgomery D. Astrocytes: from functions and role in disease. Vet Pathol. 1994;31:145–167.[Abstract]
45. Chen H, Chopp M, Schultz L, Bodzin G, Garcia J. Sequential neuronal and astrocytic changes after middle cerebral artery occlusion in the rat. J Neurol Sci. 1993;118:109–116.[Medline] [Order article via Infotrieve]
46. Garcia J, Yoshida Y, Chen H, Li Y, Zhang Z, Lian J, Chen S, Chopp M. Progression from ischemic injury to infarct following middle cerebral artery occlusion in the rat. Am J Pathol. 1993;142:623–635.[Abstract]
47. Levine S. Anoxic-ischemic encephalopathy in rats. Am J Pathol. 1960;36:1–17.
48. Pulsinelli WA, Brierly JB, Plum F. Temporal profile of neuronal damage in a model of transient forebrain ischemia. Ann Neurol. 1982;11:499–509.[Medline] [Order article via Infotrieve]
49. Feuerstein G, Wang X, Barone F. Inflammation-related gene expression and stroke: implications for new therapeutic targets. In: Krieglstein J, ed. Pharmacology of Cerebral Ischemia. Stuttgart, Germany: Medpharm Scientific Publishers; 1996:405–419.
Editorial Comment
Department of Cardiovascular Sciences, DuPont Pharmaceuticals Company, Wilmington, Delaware
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesIntroduction References |
|---|
Benjelloun et al report in the accompanying article that an extensive inflammatory reaction in response to focal ischemia evolves in the neonatal rat brain. To those involved in investigations of immune and inflammatory reactions associated with brain injury, the data come as no surprise; over the past few years, numerous reports have pointed out in a quite detailed manner that inflammation and possibly immune components are markedly activated de novo in brain tissue injured by trauma, ischemia, toxins, and infectious agents.1 2 3 The report by Benjelloun et al extends the subject to neonatal life, as also reported by Hallenbeck and Kochanek1 and others using various trauma models. Most welcomed in the present report are (1) long-term follow-up (months); (2) the inclusion of T-cell lymphocytes in the study (this cellular component of the immune system has not been frequently studied); and (3) the association of the Bcl-2/Bax system of cell death regulatory pathways along with the inflammatory reaction. Taken together, the report provides high-quality data that amplify and strengthen the fact that immune/inflammatory cells and mediators are part of the brain response to injury. The conclusion forwarded by the authors in the abstract and further reiterated in the article is that inflammatory responses may be involved in accelerating the destructive process in perinatal ischemia. With this in mind, it is of interest to point out that the conclusion by the authors of this article, as in all previous reports, is in marked contrast to the reputable role of inflammation in tissue injury as defined by medical dictionaries, such as the Stedman's,4 in which inflammation is described as "... a dynamic complex of cytologic and chemical reactions that occur in the affected blood vessels and adjacent tissues in response to an injury or abnormal stimulation ... including: 1) the local reactions ... , 2) ... removal of the injurious material, 3) the responses that lead to repair and healing." Similar definitions can be found in textbooks of inflammation5 and pathology.6 Clearly, the sense and purpose of the inflammatory reaction to repair, reconstruct, and heal have not been favored in the discussion sections of articles that describe the cellular and biochemical components of the inflammatory reactions to brain ischemia/trauma. This discrepancy is even more perplexing in view of several important reports that demonstrate the following: (1) lack of therapeutic benefits of glucocorticosteroids or NSAIDs in stroke or head injury; (2) experimental models with genetic deletion of cytokine signaling elements (eg, tumor necrosis factor-
receptor knockout) show no protection against
ischemia or other injuries (on the contrary, a measure of
vulnerability has been noted)7 ; (3) activated
macrophages and T-cell lymphocytes are of therapeutic potential
in brain and spinal cord injury8 ; and (4) some of the most
"maligned" mediators in reference to ischemic brain injury,
interleukin-1 and tumor necrosis factor-, serve as inducers
of tolerance to certain experimental paradigms of brain
injury.9 These comments are not to be taken as intentional "contrarianism" to the prevalent dogma on the role of inflammation in brain injury; rather, in my opinion, it is time to adopt a more sophisticated understanding that immune and inflammatory cells and mediators serve complex processes, including healing elements.
We should seek to identify the cells/mediators that serve in the specific functions of the reaction, aiming for suppression of particular cells/mediators that act in specific time frames to augment injury while enhancing cells/mediators (which could be similar) that mediate healing at other phases after injury. Such views may help to define better strategies for pharmaceuticals that may not target suppression of the inflammatory reaction at large, including the use of specific agents that may support regeneration/reconstruction exemplified by many functions of the inflammatory reaction itself.
Received February 4, 1999; revision received April 29, 1999; accepted June 2, 1999.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesIntroduction References |
|---|
1. Hallenbeck JM, Kochanek PM. Inflammatory responses in cerebral ischemia: role of leukocytes. In: Ginsberg MD, Bogousslavky J, eds. Cerebrovascular Diseases. Philadelphia, Pa: Blackwell Science; 1998:489–506.
2. Feuerstein GZ, Wang X, Barone FC. Inflammatory mediators and brain injury: the role of cytokines and chemokines in stroke and CNS diseases. In: Ginsberg MD, Bogousslavky J, eds. Cerebrovascular Diseases. Philadelphia, Pa: Blackwell Science; 1998:507–531.
3. Chopp M, Zhang RL, Chen H, Li Y, Jiang N, Rusche JR. Postischemic administration of anti-Mac-1 antibody reduces ischemic cell damage after transient middle cerebral artery occlusion in rats. Stroke. 1994;25:869–876.
4. Stedman's Medical Dictionary. 26th ed. Baltimore, MD: Williams & Wilkins; 1995.
5. Fantone JC, Ward PA. Pathology. Rubin E, Farber JL, eds. 1994.
6. Gallin JI, Goldstein IM, Snyderman R. Inflammation: Basic Principals and Clinical Correlates. New York, NY: Raven Press; 1988.
7. Bruce AJ, Boling W, Kindy MS, Peschon J, Kraemer PJ, Carpenter MK, Holtsberg FW, Mattson MP. Altered neuronal and microglial responses to brain injury in mice lacking TNF receptors. Nat Med. 1996;2:788–794.[Medline] [Order article via Infotrieve]
8. Moalem G, Leibovitz-Amit R, Yoles E, Mor F, Cohen IR, Schwartz M. Autoimmune T cells protect neurons from secondary degeneration after central nervous system axotomy. Nat Med. 1999;5:49–55.[Medline] [Order article via Infotrieve]
9. Ohtsuki T, Ruetzler CA, Tasaki K, Hallenbeck JM. Interleukin-1 mediates induction of tolerance to global ischemia in gerbil hippocampal CA1 neurons. J Cereb Blood Flow Metab. 1996;16:1137–1142.[Medline] [Order article via Infotrieve]
This article has been cited by other articles:
 |
|
 |
|
  S. Villapol, A. Gelot, S. Renolleau, and C. Charriaut-Marlangue Astrocyte Responses after Neonatal Ischemia: The Yin and the Yang Neuroscientist, August 1, 2008; 14(4): 339 - 344. [Abstract] [PDF]
|
|
|
|
 |
|
 W. Yin, A. P. Signore, M. Iwai, G. Cao, Y. Gao, M. J. Johnnides, R. W. Hickey, and J. Chen Preconditioning Suppresses Inflammation in Neonatal Hypoxic Ischemia Via Akt Activation Stroke, March 1, 2007; 38(3): 1017 - 1024. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Z.-Q. Zhao and J. Vinten-Johansen Myocardial apoptosis and ischemic preconditioning Cardiovasc Res, August 15, 2002; 55(3): 438 - 455. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. H. Lee, D. G. Na, K. H. Choi, K. J. Kim, J. W. Ryoo, S. Y. Lee, and Y.-L. Suh Subcortical Low Intensity on MR Images of Meningitis, Viral Encephalitis, and Leptomeningeal Metastasis AJNR Am. J. Neuroradiol., April 1, 2002; 23(4): 535 - 542. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Shibata, S. R. Kumar, A. Amar, J. A. Fernandez, F. Hofman, J. H. Griffin, and B. V. Zlokovic Anti-Inflammatory, Antithrombotic, and Neuroprotective Effects of Activated Protein C in a Murine Model of Focal Ischemic Stroke Circulation, April 3, 2001; 103(13): 1799 - 1805. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Derugin, M. Wendland, K. Muramatsu, T. P. L. Roberts, G. Gregory, D. M. Ferriero, Z. S. Vexler, and W. D. Dietrich Evolution of Brain Injury After Transient Middle Cerebral Artery Occlusion in Neonatal Rats Editorial Comment Stroke, July 1, 2000; 31(7): 1752 - 1761. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||