Treatment with Nasal Neuro-EPO Improves the Neurological, Cognitive, and Histological State in a Gerbil Model of Focal Ischemia

Vascular illness of the brain constitutes the third cause of death and the first cause of disability in Cuba and many other countries. Presently, no medication has been registered as a neuroprotector. Neuroprotection with intranasal Neuro-EPO (EPO, erythropoietin) has emerged as a multifunctional therapy that plays a significant role in neural survival and functional recovery in an animal model of stroke. On the other hand, there is limited access to the brain through the blood brain barrier (BBB) for intravenously applied EPO, and the high EPO dosages needed to obtain a protective effect increase the danger of elevated hematocrit levels and practically exclude chronic or subchronic treatment with EPO. A promising approach has been recently developed with a nonerythropoietic variant of EPO, Neuro-EPO, with low sialic acid content, a very short plasma half-life, and without erythropoietic activity, probably similar to endogenous brain EPO. The objective of this work was to determine the neuroprotective effect of intranasal Neuro-EPO in comparison with the human recombinant EPO injected intraperitoneally in the acute phase of cerebral ischemia, employing the common carotid artery occlusion model in gerbils. Neuro-EPO has demonstrated a better neuroprotective effect, evidenced through increased viability, improvements of the neurological state and cognitive functions, as well as protection of the CA3 region of the hippocampus, temporal cortex, and the thalamus. In conclusion, the intranasal application of Neuro-EPO has a better neuroprotective effect than intraperitoneal EPO, evidenced by the significant improvement of neurological, cognitive, and histological status in the animal model of stroke employed.


INTRODUCTION
relative humidity 55-60%; light-dark cycle was 12 h/12 h and there were 15-20 room air changes per hour. All materials used to maintain the animals were autoclaved at 121°C for 20 min. Food and water was provided ad libitum. Each protocol was discussed and approved by the Institutional Ethics Committee, considering the international standards established by ICLAS [26]. The animals were distributed according to a completely randomized design in four groups of 35  IN administration was performed essentially as described previously [27], taking into account the established Guide to the Care and Use of Experimental Animals [26]. Immediately after surgery, the gerbils were placed on their backs and a total of 10 μg (249.4 UI) of Neuro-EPO or a corresponding volume of vehicle solution per gerbil was given in nose drops (5 μl/drop) over a 1-to 2-min period, alternating drops between the left and right nares. The mouth and the opposite naris were closed during the administration so the drops could be naturally inhaled high into the nasal cavity. Neuro-EPO IN, a corresponding volume of vehicle IN, and EPO IP total dose 5000 UI EPO were administered three times per day during 4 days, beginning immediately after surgery.

Surgical Procedure and Spontaneous Exploratory Activity Measurement
Gerbils were anesthetized with ketamine-atropine-diazepam (47, 0.02, and 5 mg/kg, respectively). Lesions were performed according to Butterfield and McGraw's method [28]. Briefly, the right common carotid artery (CCA) was isolated, double ligated using silk 5-0 suture, and sectioned. In control operated animals, the artery was only isolated.
Twenty-four hours after unilateral permanent ischemia, the appearance of the following clinical signs of infarction was assessed: palpebral ptosis, bristling, loss of tone and reflexes in the four limbs, postural asymmetry, rolling or circling, and death. Each sign was scored separately. The sum of scores creates a general neurological score for each animal [29]. The clinical signs of brain infarction were assessed as previously established [27]. In brief, each animal was evaluated to determine its neurological state according to a scale (with a maximum of 30 and a minimum of 0), grasping strength, and disturbances in posture and gait.
Gerbils were placed in the center of a round open field (30 cm diameter and 25 cm high). "Rearing" was considered as standing straight up on the hind limbs and tail, until the animal returned any forelimb to the floor or touched the open field wall with any forelimb. Neither a subsequent straight after a rearing without reaching the floor with any forepaw nor kangaroo-like posture was considered. Exploratory activity was determined by the rearing counted at 3, 6, and 9 min in the open field. An average of the total rearing counts was calculated for each group, rendering three dots per trial. Dots were plotted, and the line obtained by the minimal square method was calculated and considered as the habituation curve. The slope of the habituation curve was used to characterize the animal's state.

Tissue Preparation
 Histology: gerbils were anesthetized for cardiac perfusion. Each animal was perfused with 20 ml of saline solution and fixed with 60 ml of 4% buffered formaldehyde solution, pH 7.0. Brains were carefully removed and postfixed in the same solution for several days. Fixed brains were dehydrated and embedded in paraffin. Serial coronal sections of 8-μm width were obtained. Sections were stained with hematoxilin-eosin and described using light microscopy.  Semi-quantitative analysis: a semi-quantitative scale was constructed to evaluate the histological state of each region. The following regions were considered in each hemisphere: hippocampus (CA1, CA2, CA3, CA4 regions), thalamus, caudate-putamen, parietal and temporal cortex. According to the histological aspect, each region was assigned between 0 and 3 points, equivalent to no damage (0), slight (1), moderate (2), and severe (3). The score attained by each animal was denominated grade histology (GH). An animal could reach a maximum GH of 21 and a minimum of 0 for each hemisphere.  Quantitative morphological analysis: preparations containing dorsal hippocampus (from 1.3 to 1.6 after Bregma), according to the atlas by Loskota et al. [30], were used. Observers were blind to the preparation origin. Pictures of 400 magnifying power were digitized and processed by the ImageJ program (http://rsb.info.nih.gov/ij/, NIH). The area of hippocampus CA1 pyramidal layer was measured, subtracting the area occupied by pyknotic cells. Then, the normal remaining area of CA 1 subfields was determined.

Statistical Analysis
The log rank test using GraphPad Prism 4 to evaluate survival percentage for the three treated groups and control was employed. Frequencies of clinical signs and rearing counts were analyzed employing the Chisquare independence test. The Mann-Whitney U test was used for comparisons between the control and others groups of neurological score rearing counts, slope, and remaining CA1 area. The Wilcoxon matched pair test was used for comparisons between hemispheres. For correlations between morphological and functional variables, the Spearman's rank correlation coefficient was used. The statistical analysis was carried out according to the Microsoft STATISTICA version 6.0 program. In all, a level of significance of p < 0.05 was accepted.

Survival
Survival percentage for the three treated groups was significantly lower than for the control group, but the survival of the group treated with Neuro-EPO IN was higher than that of animals treated with vehicle and EPO IP, although comparison with the latter did not achieve statistical significance (Fig. 1).

Clinical Signs of Stroke
The neurological score in the treated groups (Neuro-EPO IN and EPO IP) was significantly smaller than in the vehicle group, but there was no difference between the two treated groups (Fig. 2)

Spontaneous Exploratory Activity
Analyzing the slopes of the habituation curves in the open field test (Fig. 3), it could be established that in all the experimental groups, except the group tested with the vehicle, significant differences existed between the habituation curves before and after carotid occlusion. These differences in the control group and in the group treated with Neuro-EPO IN were positive (greater slope after the operation), indicating that these animals were able to recognize the field, while in the groups treated with the vehicle and EPO IP, it was negative (smaller slope in the habituation curve after the operation), indicating a lack of recognition of the previously explored field.

Qualitative Study
The control group showed no histological lesions of cerebral ischemia in the regions studied. In the ischemic group treated with the vehicle, a large stroke area was observed in the hemisphere ipsolateral to the occlusion of the carotid at 7 days after injury. The stroke area was identified by the death of most of the cellular elements, and presence of cellular remains and of partially empty areas. The stroke area extended to all the regions of the hippocampus, to the parietal and temporal cortex, to nuclei of the thalamus and caudate-putamen. In sectors CA1 and CA3 of the hippocampus, pyramidal pyknotic neurons were observed with acidophilius cytoplasm. Many cells were observed in cariorrexis processes, cariolisis and others in a state of "ghost cells." In the stroke area, full granulate-greasy corpuscles with products of degradation of myelin were observed. In the caudate-putamen nuclei of both hemispheres, extensive areas of stroke were observed with the presence of abundant heaps of cells, polyploidy, processes in mitosis, also absence of the neuropil and of all the cellular elements.
In the ischemic group treated with EPO IP, irreversible damage in the CA1 and CA2 regions of the hippocampus was detected, and these lesions were characterized by selective neuronal death and increase in the number of microglía. The pyramidal neurons of the hippocampus were eosinophilic and the existence of empty spaces revealed possible neuronal loss. Protection of the CA3 region of the hippocampus was observed. Stroke areas were observed in the parietal cortex and caudate-putamen nucleus, with protection of the histological structure of the temporal cortex. Cell polyploidy and processes in mitosis were observed in the thalamus (Figs. 4 and 5).
In the ischemic group treated with Neuro-EPO IN, there was slight damage in the CA1 and CA2 regions of the hippocampus, while in the CA3 region, the pyramidal neurons remained normal; isolated areas of stroke were observed in the parietal cortex, with slight pyknosis. In the temporal cortex and thalamus, conservation of the histological structures prevailed in all the layers. The stroke area was less extensive in the ischemic group treated with Neuro-EPO IN than in the ischemic animals treated with EPO IP and with the vehicle (Figs. 4 and 5).

Semiquantitative Analysis
Semiquantitative evaluation of the histological scale showed that animals treated with Neuro-EPO IN displayed a lower histological grade than the ischemic animals treated with the vehicle, fundamentally at the expense of CA3, thalamus, and temporal cortex of the ipsilateral hemisphere. On the other hand, animals treated with EPO IP had a significantly lower histological grade in the CA3 region and temporal cortex with respect to those treated with the vehicle, not finding significant differences with any other region (Table 1).

Quantitative Analysis
The number of healthy neurons in the contralateral hemispheres (left) was the same in the four groups (p > 0.05). In the hemisphere ipsilateral to the occlusion, the number of healthy neurons in CA1 was significantly smaller in the groups where the carotid was injured, i.e., in those treated with vehicle, Neuro-EPO IN, and EPO IP, than in the control group (Table 2).

DISCUSSION
The present study is the first to investigate the effect of Neuro-EPO IN vs. EPO IP on behavioral and histological variables in focal ischemia in gerbils. Previous to this work, only a few publications appeared in MEDLINE evaluating rHu-EPO in vivo as a neuroprotector in cerebral ischemia models in gerbils [31,32,33,34,35]. The routes employed to deliver EPO to the damaged brain were IP [33], ICV [34,36], and/or IP-ICV [37].   Nasal delivery of therapeutic proteins to the CNS clearly involves extraneuronal transport as it occurs in minutes rather than hours. It is an attractive possibility to use in acute stroke where "time is brain". In a previous paper, we showed that Neuro-EPO IN reached the CNS in pharmacological concentration in only 5 min [27]. This result in rodents was confirmed with a pharmacokinetic study in nonhuman primates with IN application of Neuro-EPO. In this same study, the IN administration of Neuro-EPO resulted in 3.5 times higher concentration of CSF EPO levels of times faster that the administration of IV rHu-EPO [23]. We previously demonstrated the presence of I125-labeled Neuro-EPO in the brain after an intranasal single-dose application, strongly suggesting the passage of the molecule to the brain regions not anatomically related [27,38]. This supports the findings of other authors and our previous reports on the neuroprotector effect of applied IN EPO in stroke models [16,21]. Mortality in this model of stroke reflects the intensity of the lesion and it is an important indicator for the evaluation of drugs in the treatment of ischemia. For this model, rates of mortality between 30 and 40% at 24 or 48 h from the lesion, respectively, have been reported [39,40], agreeing with our results. Survival of the group treated with Neuro-EPO IN in this work was higher than that of animals treated with the vehicle and EPO IP, confirming our previous reports on the neuroprotective effect of Neuro- EPO  IN[24,27,41].
To assess the mechanisms through which nasal Neuro-EPO achieves its neuroprotective effect is beyond the scope of this work, but we speculate that they are probably related toh the same mechanisms described for rHu-EPO or its derivatives CEPO or sialo EPO in different models of stroke: antiapoptotic [42,43,44,45], anti-inflammatory [46,47], neurogenic [48,49], angiogenic [15,50,51,52], and antioxidant [53,54,55,56]. Further animal experiments of Neuro-EPO IN on focal ischemia models are necessary in order to understand the mechanisms underlying improved survival.
Animals with more severe clinical signs of stroke corresponded with two groups: vehicle and EPO IP. In these cases, the remarkable lack of rearing in a typical active rodent is due to motor, rather than to other behavioral impairments. According to open field performance, these groups had a significantly depressed exploratory activity compared to Neuro-EPO IN before and after 7 days of ischemia and treatments.
These results suggest the existence of a subtle functional alteration in this subset of animals, probably at the synaptic level, undetectable with hematoxylin-eosin staining.
The histological analysis demonstrated that Neuro-EPO IN produced neuroprotection in the CA3 region of the hippocampus, thalamus, and temporal cortex, regions that constitute the frontiers of stroke, and mainly in the region of the thalamus, while the application of EPO IP did not. Neuro-EPO IN could be related with the improved learning found in the open field test.
According to the open field test, we conclude that the animals treated with vehicle and with EPO IP present cognitive dysfunction associated with ischemia and especially to the CA1 sector of the hippocampus that was seriously damaged, the most vulnerable area to ischemic damage, which regulates and controls learning and memory. These animals do not remember the environment explored 7 days after having explored it for the first time, thus they continue zooming with the same frequency in the three intervals of time. However, the animals treated with Neuro-EPO IN present a smaller damage, which does not affect their learning or memory.
Other authors demonstrated that ICV administration of EPO increased the natatorium capacity, and diminished the area of cortical stroke and thalamic degeneration in rats with permanent focal ischemia [37]. In our work, the structures where neuroprotection was observed were CA3, thalamus, and temporal cortex localized at the periphery of the stroke area, and probably constituted the area of ischemic penumbra during the first hours after the occlusion. Previous results with Neuro-EPO IN in medial cerebral artery occlusion show a significant reduction in the infarcted area in animals subjected to focal ischemia compared with animals treated with the vehicle of the formulation and rHu-EPO IP [38].
It is considered that in the ischemic penumbra area, a blood flow below normal levels still exists [57]. Probably, the arrival of Neuro-EPO to the region of ischemic penumbra protected those cells against phenomena like excitotoxicity and apoptosis [14]. Also, it has been demonstrated that EPO stimulates angiogenic and neurogenic processes [11,15,58], providing a favorable microenvironment for neuronal plasticity during the recovery from stroke. In this work, during the 7 days of ischemia, histological damage was diminished in the above-mentioned areas.
In the CA1 region of the hippocampus, good neuroprotection was not observed, a finding corroborated with neuron counting. This area of the hippocampus is the most sensitive to ischemia [59], for which the neuroprotective effect of any drug would have lower possibilities of being evidenced than in less vulnerable areas. Animals from the ischemic group treated with the vehicle presented necrosis of the parietal and temporal cortex, hippocampus, thalamus, and caudate-putamen; loss of pyramidal neurons of CA1; and cognitive deterioration; findings similar to those described in other works [60,61]. These negative effects were reduced by the pharmacological effect of Neuro-EPO IN.
The results of this work constitute an indirect evidence of the passage of Neuro-EPO to the CNS through the nasal cavity. The nasal route gives the possibility of administering much lower doses than those necessary by the IV route [16]. These results suggest that Neuro-EPO IN can have a therapeutic effect on stroke damage after focal cerebral ischemia, applying a dose that is lower than those previously reported for any recombinant EPO.
Taken together, all these results suggest that Neuro-EPO IN is a better neuroprotectant than EPO IP, at least in this model of stroke.
The therapeutic strategy of developing the nasal delivery of Neuro-EPO could allow for its multiple and chronic use in neurodegenerative disorders.
In conclusion, ours results using Neuro-EPO IN suggest that it could be a potential novel neurotherapeutic approach for the treatment of ischemic stroke. The above-mentioned studies also indicate the safety and proof-of-concept of the initiation of clinical trials in stroke patients.

DISCLOSURE
Drs. García Rodríguez and Sosa Testé are coinventors on the patent "rh-epo nasal formulations with low sialic acid concentration for the treatment of diseases of the central nervous system", cu. Patent Formulation International Application No.: PCT/CU2006/000007 OCPI, Havana, Cuba. The authors report no other conflict of interest concerning in this paper.