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Erythropoietin is neuroprotective, improves functional recovery, and reduces neuronal apoptosis and inflammation in a rodent model of experimental closed head injury

I. Yatsiv^*,, N. Grigoriadis, C. Simeonidou, P. F. Stahel, O. I. Schmidt, A. G. Alexandrovitch^*, J. Tsenter^*,|| and E. Shohami^*,1

^* Departments of Pharmacology,
Pediatric Intensive Care and
^|| Rehabilitation, Hebrew University Hadassah Medical Center, Jerusalem, Israel;
Department of Neurology AHEPA University Hospital and Department of Physiology, Faculty of Medicine, Aristotle University Thessaloniki, Greece; and
Department of Trauma and Reconstructive Surgery, Charité University Medical School, Berlin, Germany

¹Correspondence: Department of Pharmacology, School of Pharmacy, The Hebrew University of Jerusalem, Jerusalem 91120, Israel. E-mail: esty{at}cc.huji.ac.il

SPECIFIC AIMS

To date there are no pharmacological agents to treat traumatic brain injury. The present study was aimed to demonstrate the protective effect of erythropoietin (EPO) treatment after brain injury in the mouse. We assessed the effect of treatment at the neurobehavioral and cognitive level, as well as at tissue level (reduced inflammation) and cellular level (less neuronal apoptosis).

PRINCIPAL FINDINGS

1. Effect of posttraumatic rhEpo treatment on the neurobehavioral recovery
The functional recovery after injury was assessed by serial measurements of NSS in vehicle- and rhEpo-treated mice. To ensure a comparable severity of trauma in both groups, mice were assigned to the different groups after their initial NSS was evaluated, at 1 h. As shown in Fig. 1 , both groups had a similar initial NSS 1 h with a mean of 6.44 ± 0.17 and 6.50 ± 0.25 for the vehicle- and rhEpo-treated groups, respectively, indicating a similar severity of injury. Evaluation of NSS at later time points revealed faster recovery, as shown by lower mean NSS values compared with the control group. Three days after injury, functional recovery of the rhEpo-treated mice was significantly improved, as evident by a lower mean NSS values as compared with control (4.11±0.31 vs. 5.06±0.15, respectively, P<0.05, Mann-Whitney U test). The difference between the groups remained highly significant during the 14 days observation period.

View larger version (11K): [in this window] [in a new window]   Figure 1. rhEpo improves neurobehavioral recovery after closed head injury. rhEpo was administered 1 and 24 h after injury. Neurobehavioral status was evaluated by the neurological severity score (NSS), such that reduction in NSS reflects faster recovery. A beneficial effect of rhEpoin-treated vs. vehicle-treated mice was found as early as 3 days after trauma (*P=0.003) and became more significant for up to 14 days (**P=0.007, #P<0.0001).

2. Effect of post-traumatic rhEpo treatment on recovery of cognitive functions
The effect of rhEpo on post-traumatic recovery of memory was examined by applying the object recognition test (ORT). Rodents have a natural tendency to explore objects in their vicinity, so that when familiar and new objects are compared the latter attracts more attention and more time is spent exploring it. The percent of time spent at exploring a new object out of the total exploration time was calculated for vehicle- and rhEpo-treated mice. Three days after injury, vehicle-treated mice spent 48.5 ± 4.8% exploring the new object, while rhEpo-treated mice spent 71.7 ± 3.2% of the total exploration time at the new object. This difference is highly significant (P<0.001; Student’s t test), indicating that the treated mice are able to distinguish between the "memorized" and new objects. Another group of mice was tested 8 days after injury, but at this time point the difference between vehicle- and rhEpo-treated groups diminished and became nonsignificant (61.7±2.6 vs. 69.9±4.0; P=0.1; Student’s t test), probably due to the spontaneous recovery of the vehicle-treated group.

3. The effect of rhEpo treatment on neuronal apoptosis and axonal damage
Apoptotic cells were detected mainly at the acute stages (24 h) after injury, and were identified with TUNEL and supported by activated caspase-3 immunohistochemistry. The percentage of apoptotic neurons was significantly lower in the cortical areas adjacent to trauma in rhEpo- compared with vehicle-treated animals. Quantification of the data yielded 7.9 ± 0.4 vs. 27.6 ± 0.6% apoptotic cells; P = 0.033 (Fig. 2E ) in the rhEpo- and vehicle-treated mice, respectively. In the hippocampus, neuronal apoptosis was evident in most of the untreated mice 24 h after trauma, whereas none of the brain-injured rhEpo-treated animals displayed apoptosis of hippocampal neurons (not shown). When examined 24 h after injury, there was no difference between the two groups regarding the amount of axonal injury or axonal loss. However, at 7 days a marked effect of rhEpo treatment was evident. Figure 2 depicts Bielcshowski silver impregnation staining of Epo- and vehicle-treated brain-injured mice. Axons in the rhEpo-treated mice were relatively well preserved compared with vehicle-treated animals as shown by their density within the area of trauma (P<0.01) (Fig. 2E ). Thus, similar level of moderate axonal loss was found in the brains of the vehicle- and rhEpo-treated mice (grade 1), whereas in rhEpo-treated mice a significant increase in the percent of normal axonal density (grade 0, P<0.001) and a decrease in the severe axonal loss (grade 2) were found.

View larger version (71K): [in this window] [in a new window]   Figure 2. rhEpo preserves axons 7 days after closed head injury. Bielschowsky silver impregnation staining indicates severe axonal injury after TBI (B–D) compared with sham (A). Axonal injury (arrows) and loss were more evident in vehicle (B, C) compared with rhEpo (D) -treated animals. E) Bars represent number of optical fields with normal axonal density and moderate or severe axonal loss for the 2 animal groups. rhEpo-treated animals have an obvious shift of axonal pathology toward normal axonal density when compared with vehicle-treated animals. This observation is significant at the level of P < 0.001 (Chi square test). 0, normal axonal density; 1, moderate axonal loss; 2, severe axonal loss.

4. The effect of rhEpo treatment on posttraumatic neuroinflammation
Rats treated with rhEpo showed a significant recovery of function 1 wk after trauma (lower NSS; P<0.05, data not shown), then were sacrificed to evaluate the effect of rhEpo on neuroinflammation. An impressive reduction of complement activation at the C3 level is demonstrated as attenuated C3 deposition in injured tissue in rhEpo- vs. vehicle-treated rats. A clearly reduced activation of glial cells (GFAP-positive astrocytes and CR3-positive microglia, shown as CD11b positive) in the injured hemisphere of rhEpo- vs. vehicle-treated rats, is also shown.

CONCLUSIONS AND SIGNIFICANCE

The present study demonstrates that rhEpo conferred neuroprotection in an in vivo experimental model of TBI and emphasizes its beneficial effect on neurobehavioral and cognitive functions. The effect on neurobehavioral parameters was significant and lasted for >14 days. rhEpo-treated mice had significantly less apoptotic nuclei, better preservation of axons, and less inflammatory infiltrates.

The role of Epo as a neuroprotectant has been studied in in vitro and in vivo experimental models of brain injury. These include ischemia, hypoxia, kainate-induced seizures, brain and spinal cord trauma, MPTP-induced Parkinson disease, and EAE, rhEpo drug was administered either 24 h before or at various intervals (up to 6 h) after brain trauma, and animals were killed 10 days later. rhEpo-treated animals had a reduced volume of injury and a marked decrease in the number of inflammatory cells surrounding the necrotic core. The latter findings agree with our results, in which the effect on functional recovery was demonstrated. Epo and EpoR are abundant in the spinal cord, which, similar to the brain, responds favorably to Epo treatment. rhEpo administration in rat models of spinal cord compression and contusion was associated with markedly improved neurological recovery. In these rhEpo-treated rats white matter was preserved, and minimal axonal degeneration and apoptosis were found. These and similar findings agree remarkably with our current findings in experimental closed head injury model.

The effect of rhEpo on the recovery of cognitive functions after head trauma has not been studied to date, and to our knowledge this is the first report showing improved cognitive function after TBI in rhEpo-treated mice. These results support those that demonstrated preservation of learning function after rhEpo treatment in brain ischemia. Taken together, our current findings, along with earlier reports in other models, suggest a role for Epo in recovery of cognitive functions.

Effect of rhEpo on histopathology
To better understand the mechanism by which Epo provides neuroprotection, we studied the effects of rhEpo treatment on histopathology, inflammation, and apoptosis in different areas of the brain at two time points (acute, t=24 h and chronic, t=7–14 days after injury).

Inflammation and apoptosis
Accumulation of inflammatory cells into the impact site and surrounding area is well documented after trauma. This phenomenon is a cornerstone of inflammation and has been described extensively, including in the closed head injury model used in the present study. In the present study, inflammation, assessed 7 days after injury, is expressed by increased intracerebral complement deposition and activation of glial cells (expressed as GFAP and CR3-positive cells). These were markedly attenuated in the reEpo-treated animals.

Neuronal apoptosis was assessed by TUNEL staining and morphological criteria, and was confirmed with activated caspase-3 immunohistochemistry. It was mainly evident at the acute (24 h) period, when a clear decrease in apoptosis was found in cortical neurons in the rhEpo-treated animals. This finding was expected as apoptosis has been described as an inflammation-limiting mechanism that is activated when the inflammatory response in and around the trauma area has to be contained.

The effect of rhEpo on axonal injury
The effect of rhEpo on axonal injury was also studied. While in the acute phase (24 h) axonal injury or axonal loss were similar in the two groups, at 7 days postinjury, axons of the rhEpo-treated group were relatively well preserved, as shown by their density within the area of trauma (P<0.01) (Fig. 2) . This finding may indicate that axonal injury induced at the acute stages after trauma is at least partly reversible by Epo treatment. Alternatively, one can propose that rhEpo treatment reduces ongoing axonal damage at the chronic stages.

In conclusion, our present findings add to the cumulating evidence on the nonhematopoietic neuroprotective effects of Epo. In view of its long-term safe use in end-stage renal failure and oncology patients, and in view of the lack of pharmacological therapy to head trauma patients and the success of Epo treatment in clinical stroke, we believe that a clinical trial using Epo after traumatic brain injury is warranted.

View larger version (26K): [in this window] [in a new window]   Figure 3. Traumatic brain injury activates within minutes to hours several harmful pathways in the brain, including oxidative stress and inflammation. These act in concert to disrupt the blood brain barrier (BBB) integrity and to induce edema. Together, these and other pathways lead within days to weeks to necrosis, apoptosis, and axonal injury accompanied by an inflammatory response. At the functional level, the above events are expressed as impaired motor and cognitive functions, and these are the main devastating sequelae of TBI. Treatment with rhEpo at 1 and 24 h leads to reduced inflammation, apoptosis, and axonal injury. The functional manifestation of these effects was expressed as reduction in the neurobehavioral severity score and better performance in the cognitive test.

FOOTNOTES

To read the full text of this article, go to http://fasebj.org/cgi/doi/10.1096/fj.05-3907fje;

This article has been cited by other articles:

				L. Cherian, J. C. Goodman, and C. Robertson Neuroprotection with Erythropoietin Administration Following Controlled Cortical Impact Injury in Rats J. Pharmacol. Exp. Ther., August 1, 2007; 322(2): 789 - 794. [Abstract] [Full Text] [PDF]

This Article Full Text (PDF) All Versions of this Article: 19/12/1701 05-3907fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Yatsiv, I. Articles by Shohami, E. Search for Related Content PubMed PubMed Citation Articles by Yatsiv, I. Articles by Shohami, E.