Therapeutic effects of umbilical cord blood plasma in a rat model of acute ischemic stroke – europe pmc article – europe pmc fetal anoxia

Umbilical cord blood plasma (UCB-PL) contains various cytokines, growth factors, and immune modulatory factors that regulate the proliferation and function of immune cells and adult stem cells. Despite its therapeutic potential, the effects of UCB-PL treatment in conditions of ischemic brain injury have yet to be investigated. In this study, we demonstrated that both behavioral and structural impairments resulting from ischemic brain injury were significantly prevented/reversed after intravenous administration of UCB-PL relative to the vehicle control. As early as 1-week post-ischemia, an increased number of newborn cells in the subventricular zone and a reduced number of activated microglial cells in the peri-infarct area were observed in the UCB-PL group, suggesting that enhanced neurogenesis and/or the suppression of inflammation may have contributed to functional protection/recovery.Fetal anoxia


moreover, UCB-PL was more effective than plasma derived from a 65-year-old healthy adult for the treatment of ischemia-related structural and functional deficits, indicating that UCB-PL had greater therapeutic potential. This study provides valuable insights into the development of a safe, effective, and cell-free strategy for the treatment of ischemic brain damage and a much-needed alternative for patients who are ineligible for thrombolytic therapy.

Umbilical cord blood plasma (UCB-PL) contains various cytokines, growth factors, and immune modulatory factors that regulate the proliferation and function of immune cells and adult stem cells. Despite its therapeutic potential, the effects of UCB-PL treatment in conditions of ischemic brain injury have yet to be investigated.Fetal anoxia in this study, we demonstrated that both behavioral and structural impairments resulting from ischemic brain injury were significantly prevented/reversed after intravenous administration of UCB-PL relative to the vehicle control. As early as 1-week post-ischemia, an increased number of newborn cells in the subventricular zone and a reduced number of activated microglial cells in the peri-infarct area were observed in the UCB-PL group, suggesting that enhanced neurogenesis and/or the suppression of inflammation may have contributed to functional protection/recovery. Moreover, UCB-PL was more effective than plasma derived from a 65-year-old healthy adult for the treatment of ischemia-related structural and functional deficits, indicating that UCB-PL had greater therapeutic potential.Fetal anoxia this study provides valuable insights into the development of a safe, effective, and cell-free strategy for the treatment of ischemic brain damage and a much-needed alternative for patients who are ineligible for thrombolytic therapy.

INTRODUCTION

Stroke, a devastating cerebrovascular disease, is classified into ischemic and hemorrhagic subtypes based on its etiology [ 1, 2]. More than three-quarters of all cases are reported to result from ischemic injury. Thrombolytic therapy is the first line therapy for ischemic stroke. However, the window of effectiveness of tissue plasminogen activator (tpa) treatment is only 4.5 h post-ischemic injury, which allows only 8% of stroke patients to receive tpa therapy [ 3, 4].Fetal anoxia new cell-based therapeutic strategies are in development that use bone marrow- or adipose tissue-derived mesenchymal stem cells (mscs) and neural stem cells. However, in the case of stem cell administration to patients, the potential risk of serious side effects such as uncontrolled growth of the transplanted cells [ 5] and clogging of small blood vessels by occasional unexpected cell aggregates cannot be avoided. Therefore, from a clinical safety perspective, cell-free therapeutic strategies are more desirable if comparable efficacy can be achieved. Although conditioned media of mscs were previously tested as cell-free therapeutic strategies [ 6, 7], the exposure to fetal bovine serum during the initial establishment and expansion of the mscs raised concerns of xenopathogen transmission.Fetal anoxia

Umbilical cord blood contains a large number of stem cells (i.E., mscs and hematopoietic stem cells). Human umbilical cord blood plasma (UCB-PL) contains a variety of cytokines, growth factors, and immune modulatory factors that affect the proliferation and function of immune cells and mscs [ 8– 11]. Given the beneficial constituents of UCB-PL, it may have therapeutic utility for the treatment of a variety of disease states. Furthermore, no risk of xenopathogen transmission is associated with therapies that use UCB-PL.

In this study, we demonstrate that UCB-PL administration exerted therapeutic effects on both structural and behavioral recovery in a rat model of focal ischemic stroke. The results of this study provide valuable insights into the development of a safe, alternative, and cell-free therapeutic strategy for stroke patients who are ineligible for thrombolytic therapy.Fetal anoxia

Comparison of therapeutic effects among the UCB-PL, aged-PL, and control groups

With respect to the mnss, the UCB-PL group displayed better recovery than both the aged-PL and control groups. The difference was statistically significant at 1 week after MCAO surgery (figure ​ (figure6b, 6B, left). In the beam balance test, which is a sensitive motor assay, the UCB-PL group showed more significant improvements in behavioral performance than the aged-PL or control groups from 1 week post-MCAO and thereafter (figure ​ (figure6b, 6B, right).

Nissl staining of brain sections harvested at 3 weeks post-MCAO demonstrated that the UCB-PL group displayed a lesser degree of brain damage than the aged-PL group (24.0% in the UCB-PL group [ n = 7] vs. 63.0% in the aged-PL group [ n = 7], p 0.05) (figure ​ (figure6c 6C).Fetal anoxia

When rat brain sections were immunostained for ED1, a marker for activated microglia, and GFAP, a marker for activated astrocytes, a 44.5% reduction in the number of ED1+ cells (per 400 mm 2) and a 26% reduction in GFAP+ area (as measured by optical density) were noted near the peri-infarct striatum in the UCB-PL group compared with the aged-PL group (figure ​ (figure6d 6D).

Taken together, our results indicate that UCB-PL administration in the acute phase of an ischemic stroke model prevented or reversed behavioral and functional impairments more efficiently than aged-PL or PBS administration. Therefore, UCB-PL is a novel and promising cell-free therapeutic strategy for the safe allogeneic treatment of ischemic brain injury.Fetal anoxia

DISCUSSION

In this study, we successfully demonstrated that intravenous administration of UCB-PL improved behavioral performance post-MCAO in 3 behavioral tests, including the mnss, the corner turn test, and the foot fault test. In support of the observed functional improvements, brain infarct size as measured by both MRI and nissl staining was significantly reduced in the UCB-PL group. Therefore, our results indicate that intravenous administration of UCB-PL in the acute phase after MCAO in rats improved behavioral performance and reduced structural damage.

UCB-PL is known to contain a variety of soluble factors, including growth factors, cytokines, and immune modulatory factors [ 16– 20], that have the potential to ameliorate symptoms caused by ischemic stroke.Fetal anoxia UCB-PL, which does not contain any cells, has several advantages as a new therapeutic option; first, there is no risk of tumor formation or cell aggregation. In addition, UCB-PL is convenient to obtain, store, handle, and use (i.E., it can be frozen, thawed, transported, and administered to patients with ease) for clinical application. Furthermore, unlike bone marrow or peripheral blood, the absence of immune cells in UCB-PL signifies a decreased possibility of graft vs. Host disease (gvhd) or immune rejection.

At least possible 3 mechanisms for the efficacy of UCB-PL administration are supported by this study; (1) UCB-PL administration reduced neuroinflammation, as demonstrated by a reduced number of activated microglia in the peri-infarct area following UCB-PL administration; (2) UCB-PL administration increased neurogenesis in the SVZ area and, most likely, the migration of new cells to the injured area; and (3) soluble factors in UCB-PL had a paracrine effect on the injury site and caused the observed beneficial effects.Fetal anoxia previous studies have reported that inflammation confers detrimental effects on neural proliferation in the injured brain [ 21, 22]. Therefore, increased neurogenesis by UCB-PL administration may be indirectly supported through the suppression of microglial activation and/or directly supported by the induction of neurogenesis by soluble factors in UCB-PL.

Intriguingly, plasma from a 65-year-old healthy subject did not produce the same beneficial effects as UCB-PL, suggesting that the composition of soluble factors in plasma from UCB and aged individuals are not only different but also have different degrees of therapeutic utility. Although beyond the scope of this study, it would be of interest to specifically identify the component(s) of UCB-PL that are responsible for the observed therapeutic effects (for example, by comparing the differences in the composition of the 2 different plasmas).Fetal anoxia

Animals

This section of the study was approved by the CHA university institutional animal care and use committee. Middle cerebral artery occlusion (MCAO) was used to generate a rat model of ischemic stroke [ 23]. The surgery was conducted on adult male sprague-dawley rats ( n = 30 per group) (orient bio, seongnam, korea) weighing 240–270 g each. Briefly, rats were anesthetized with 5% isoflurane (hana pharm, hwasung, korea) and were maintained under anesthesia with 2% isoflurane in a mixture of 70% N 2 and 30% O 2. The common carotid artery, internal carotid artery (ICA), and external carotid artery (ECA) were exposed by a cervical incision, and the pterygo-palatine and occipital arteries were cauterized.Fetal anoxia blood flow was temporarily occluded by ligating the ICA and ECA. A 23-mm-long 4–0 nylon monofilament (ailee, busan, korea) coated with low-viscosity silicone (handae chemicals, chungbuk, korea) was advanced from the exposed ECA to the ICA to occlude the middle cerebral artery. Reperfusion was induced by withdrawing the suture from the ECA at 1.5 h post-ischemia.

Behavioral tests

The rats that showed similar behavioral defects were assigned randomly to each group. For behavioral assessments, each rat was subjected to a battery of behavioral tests and evaluated by an experimenter who was blinded to the treatment conditions. The modified neurologic severity score (mnss), foot fault test, and beam balance test were performed on days −3, 0, 1, 3, 5, 7, 14, and 21 post-MCAO surgery.Fetal anoxia the mnss was used to assess motor (muscle status and abnormal movement), sensory (visual, tactile, and proprioceptive), and reflex (pinna, corneal, and startle) functions [ 24]. The test results were indexed as a grade that ranged from 0 to 18 (0, no impairment; 1–6, mild impairment; 7–12, moderate impairment; and 13–18, severe impairment).

The foot fault test was performed by placing rats on a wire grate (50 cm × 50 cm with 2.5 cm × 2.5 cm grids) for 5 minutes, and the number of times the forelimbs slipped through the grate during a 5-minute session was recorded (by videotaping from below the grate) [ 25]. Raw numbers were converted to foot fault scores as follows:

Foot fault score = (number of faults for the affected forelimb/number of footsteps of the affected forelimb) − (number of faults for the unaffected forelimb/number of footsteps of the unaffected forelimb).Fetal anoxia

The beam balance test was performed by placing rats at the center of a beam (100 cm × 5 cm × 2 cm) and grading motor performance on a 6-point scale, as follows [ 26]. 1, balances with steady posture and paws on top of the beam; 2, grasps the sides of the beam and has shaky movement; 3, one or more paw slips off of the beam; 4, attempts to balance on the beam but falls off; 5, drapes over the beam but falls off; and 6, falls off of the beam without attempting to balance.

5-bromo-2-deoxyuridine (brdu) labeling

BrdU (50 mg/kg in saline) (sigma-aldrich, st. Louis, MO, USA) was administered intraperitoneally (i.P.) daily for 5 days starting the day after MCAO surgery. Rats were euthanized with a mixture of ketamine (50 mg/kg, i.P.) (huons, seoul, korea) and xylazine (5 mg/kg, i.P.) (bayer korea, seoul, korea) and transcardially perfused with 0.1 M PBS followed by 4% formaldehyde (samchun chemicals, pyeongtaek, korea).Fetal anoxia brains were removed and post-fixed in 4% formaldehyde overnight at 4°C and then sequentially cryoprotected in 15% and 30% sucrose (sigma-aldrich) in phosphate-buffered saline. Brains were then stored at −80°C in an optimal cutting temperature (OCT) solution (sakura finetek, torrance, CA, USA) and finally sectioned at a thickness of 30 μm using a cryostat (microm, HM 525, walldorf, germany). To perform brdu staining, brain sections were incubated in 2 N hcl (samchun chemicals) at 37°C for 30 min and subsequently neutralized in 0.1 M sodium borate (ph 8) (sigma-aldrich) for 20 min. Sections were then blocked with 10% goat serum (vector laboratories, burlingame, CA, USA) and 0.1% NP-40 (sigma-aldrich) in tris-buffered saline (TBS) (sigma-aldrich) for 1 h, followed by incubation with 2 μg/ml of a rat monoclonal anti-brdu antibody (abcam) at 4°C for 12 h, and then with 2 μg/ml of a goat alexa 594-conjugated anti-rat igg antibody (invitrogen) for 40 min at room temperature.Fetal anoxia sections were finally mounted on glass slides (marienfeld, lauda-koenig-shofen, germany) and examined with an LSM510 or LSM610 confocal microscope (carl zeiss, göttingen, germany).