A new model of transient focal cerebral ischemia for inducing selective neuronal necrosis
Authors: Ethem Murat Arsava
Yasemin Gursoy-Ozdemir
Hulya Karatas
Gunfer Gurer
Turgay Dalkara
Journal: Brain Research Bulletin (2009)
Discipline: NEUROSCIENCE & BEHAVIOR
Keywords: cerebral ischemia; selective neuronal necrosis; pathophysiology; transient focal ischemia; mouse model
Affiliations: Department of Neurology, Faculty of Medicine, Hacettepe University, Ankara, Turkey.

Abstract:  Brief cerebral ischemia leads to selective neuronal necrosis (SNN), which is characterized by neuronal death with sparing of glial and vascular elements of the central nervous system. Understanding the pathophysiology of SNN may help elucidating the mechanisms and consequences of neuronal injury in humans following brief ischemia. Contrary to the presence of reproducible models of transient global ischemia, animal models of transient focal ischemia producing SNN are scarce and have important limitations such as causing ischemia in a vast area and inducing additional insults. In this study, we developed a practical mouse model of SNN without these limitations, by compressing the distal middle cerebral artery (MCA) with a blunted micropipette for 15 min. The success of compression was evaluated by monitoring the regional cerebral blood flow, and conventional histopathology and immunolabeling of the brain sections. Seven/fourteen days after ischemia, intracisternally administered propidium iodide labeled numerous necrotic cells in the frontoparietal cortex, which were mostly NeuN-positive, but were not immunolabeled with astrocytic markers (GFAP and S100), and showed neuronal morphology with hematoxylin-eosin staining, indicating that the model successfully induced ischemic injury limited to neurons. The model could become an important tool for investigating the long-term effects of brief ischemic events like transient ischemic attacks and could offer convenient reversible distal MCA occlusion for studies using intravital microscopy. 

Retracted: No

PubPeer comments:
 Could the authors please check Figure 3? * Orange boxes: In the PI column, the 3rd and 4th panels down appear to look the same * Cyan boxes: In the H E column, the 3rd and 4th panels down appear to look the same The Merged images and the H E insets are different, suggesting that these might have been duplicated in error. ![file] , 
 #1 As explained in the Methods, Legend and Results of the paper, PI and H E panels on the 3rd and 4th rows are the same images except showing different areas of the same panel in insets (so, insets look different). The 3rd row illustrates co-localization of the red channel signal (PI) with green channel (GFAP) and tissue histology (H E), whereas the 4th row additionally shows co-localization of the red channel (PI) with the u201cDark Field (DF)u201d image as well as tissue histology (H E) in exactly the same brain area, processed and imaged sequentially. Insets in the 3rd and 4th rows are taken from different regions on the same panel; the one on the 4th row was marked with an arrow and arrowhead. Insets were chosen from different sets of PI-positive cells in the same panel to illustrate the phenotypic differences [e.g. GFAP-negativity, selective neuronal necrosis (SNN) morphology and occasional chromatin clumping]. Methods (page 228): NeuN, GFAP and S100 positivity were assessed with appropriate filter sets, in regions where PI positivity was observed. The same sections were also evaluated by dark-field (DF) microscopy in order to detect basophilic nuclear chromatin clumping in cells undergoing apoptosis. After the photographs were obtained from PI and either of NeuN, GFAP or S100 immunolabeled slides, hematoxylinu2013eosin staining (H E) was performed to evaluate SNN by conventional histopathology. Legend (page 229): Panels in each row show the same area of ischemic tissue sequentially processed for each staining and photographed by using the initial coordinates on microscopic slidesu2026 No colocalization of PI positive cells with u2026.. GFAP (third row) was detected. The fourth row illustrates that majority of cells with SNN lost their cell membrane integrity and hence became permeant to PI, whereas a smaller proportion of cells with SNN were PI negative but displayed apoptotic type chromatin clumping as detected by DF microscopy. A few neurons exhibited both phenotypes (arrowhead). Insets are enlarged images from the respective panels. (Note: There is a minor shift in H E micrographs that does not hinder correct tracing of the corresponding cells in other panels because it was later processed and photographed following the labeled coordinates on microscopic slides used for fluorescent imaging) Results (page 229): When the same sections were subjected to H E staining, PI positive cells that were not labeled with any of the above-mentioned antibodies showed neuronal morphology. The majority of the cells with SNN morphology were PI positive (79.1 u00b1 8.9%), whereas chromatin clumping was present in 16.7 u00b1 6.0% of these cells. Both of these features were co-localized in 4.3 u00b1 2.0% of the SNN population (Fig. 3, row 4). , 
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