Journal of Neuroimmunology 367 (2022) 577874 Contents lists available at ScienceDirect Journal of Neuroimmunology journal homepage: www.elsevier.com/locate/jneuroim Lesion-associated microglia and macrophages mediate corralling and react with massive phagocytosis for debris clearance and wound healing after LPS-induced dopaminergic depletion P. Heman-Bozadas a, b, C. Romero a, P. Martínez-Remedios a, 1, I. Freitag a, 1, A. Frías a, E. Saavedra-López a, P.V. Casanova a, M. Roig-Martínez a, G.P. Cribaro a, M.J. Rovirosa-Hernández b, D. Hernandez-Baltazar b, c, C. Barcia a, * a Institut de Neurociències & Department of Biochemistry and Molecular Biology, School of Medicine & School of Biosciences, Universitat Autònoma de Barcelona, Barcelona, Spain b Instituto de Neuroetología, Universidad Veracruzana, Xalapa, Veracruz, Mexico c Cátedras CONACyT, Dirección Adjunta de Desarrollo Tecnológico e Innovación, Consejo Nacional de Ciencia y Tecnología. CDMX, Mexico A R T I C L E I N F O A B S T R A C T Keywords: Neuroinflammation contributes to neuronal degeneration in Parkinson’s disease (PD). However, how brain in­ Parkinson’s disease flammatory factors mediate the progression of neurodegeneration is still poorly understood. Experimental Microglia models of PD have shed light on the understanding of this phenomenon, but the exploration of inflammation- Macrophages driven models is necessary to better characterize this aspect of the disorder. The use of lipopolysaccharide Lipopolysaccharide Phagocytosis (LPS) to induce a neuroinflammation-mediated neuronal loss is useful to induce reliable elimination of dopa­ Dopamine minergic neurons. Nevertheless, how this model parallels the PD-like neuroinflammation is uncertain. In the Neurodegeneration present work, we used the direct LPS injection as a model inductor to eliminate dopaminergic neurons of the Experimental models substantia nigra pars compacta (SNpc) in rats and reevaluated the inflammatory reaction. High-resolution 3D histological examination revealed that, although LPS induced a reliable elimination of SNpc dopaminergic neurons, it also generated a massive inflammatory response. This inflammation-mediated injury was charac­ terized by corralling, a damaged parenchyma occupied by a vast population of lesion-associated microglia and macrophages (LAMMs) undertaking wound compaction and scar formation, surrounded by highly reactive as­ trocytes. LAMMs tiled the entire lesion and engaged in long-standing phagocytic activity to resolve the injury. Additionally, modeling LPS inflammation in a cell culture system helped to understand the role of phagocytosis and cytotoxicity in the initial phases of dopaminergic degeneration and indicated that LAMM-mediated toxicity and phagocytosis coexist during LPS-mediated dopaminergic elimination. However, this type of severe inflammatory-mediated injury, and subsequent resolution appear to be different from the ageing-related PD scenario where the architectural structure of the parenchyma is mostly preserved. Thus, the necessity to explore new experimental models to properly mimic the inflammatory compound observed in PD degeneration. 1. Introduction questions remain unanswered regarding the particular role of inflam­ matory mediators on dopaminergic neurons (Hirsch and Standaert, Parkinson’s disease (PD) is characterized by a dramatic loss of 2020). Thus, understanding neuroinflammatory response in PD is dopaminergic neurons of the substantia nigra pars compacta (SNpc) essential to design alternative therapeutic interventions (Charvin et al., (Kalia and Lang, 2015; Przedborski, 2017). The cause of this neuro­ 2018; Wang et al., 2015). For this to be elucidated, experimental models degeneration is still under scrutiny. Although the contribution of glial- of parkinsonian-like neuroinflammation are necessary to understand the mediated neuroinflammation is now generally accepted, many action of local glial cells, as well as potential infiltrated immune cells, on * Corresponding author at: Department of Biochemistry and Molecular Biology, Institut de Neurociències, School of Medicine, Lab M2-107, Office M2-117, Universitat Autònoma de Barcelona, Bellaterra 08193, Barcelona, Spain. E-mail address: carlos.barcia@uab.es (C. Barcia). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.jneuroim.2022.577874 Received 8 June 2021; Received in revised form 17 March 2022; Accepted 19 April 2022 Available online 22 April 2022 0165-5728/© 2022 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by- nc-nd/4.0/). P. Heman-Bozadas et al. Journal of Neuroimmunology 367 (2022) 577874 neurons (Deng et al., 2020). non-dopaminergic neuronal loss seemed to be slightly higher, suggesting The SNpc of PD patients display several hallmarks of active inflam­ a progression of the neuronal degeneration. matory response such as microglial activation (McGeer et al., 1988) and Most importantly, LPS-induced dopaminergic neuronal loss was reactive astrogliosis (Forno et al., 1992) in a relatively well preserved associated with a massive accumulation of lesion-associated microglia structure of the parenchyma. Well-established neurotoxic experimental and macrophages (LAMMs), evidenced by the marker OX42 at the in­ PD models, such as MPTP or 6-OHDA (Akiyama and McGeer, 1989; jection site (Fig. 2A, B). Reduction of dopaminergic area on the ipsi­ Barcia et al., 2011; Czlonkowska et al., 1996), recapitulate the neuro­ lateral side was concomitant with the populating of a vast amount of inflammatory reaction observed in PD patients (McGeer et al., 1988), OX42+ cells compared with the contralateral side (Fig. 2B). This phe­ analogously to the incidentally intoxicated human MPTP cases (Lang­ nomenon could be seen in LPS-injected areas both at three and seven ston et al., 1999). However, in these models, the effects of the neuro- days after injection, whereas only scant levels of activation could be seen inflammatory response and neurotoxicity are hardly separable. For after saline injection (Fig. 2C). Higher resolution images of the OX42+ this reason, alternative models considering solely the inflammatory cells in the SNpc revealed drastic morphological changes in LPS-injected response are critical to understand its potential contribution to dopa­ parenchyma (Fig. 2C). Quantification of the number of OX42+ cells in minergic degeneration (Tufekci et al., 2011). the SNpc shown a dramatic increase in LAMMs in the site of injection The gram-negative bacterial membrane compound lipopolysaccha­ three days after LPS delivery. Moreover, this increase persisted at seven ride (LPS) has long been used to promote neuroinflammatory responses days, together with a mild increase observed on the contralateral side in vivo (Brown, 2019; Catorce and Gevorkian, 2016; Hoogland et al., (Fig. 2D). Skeletonized OX42+ cells from high-resolution images illus­ 2015) and in vitro (Orihuela et al., 2016; Schmid et al., 2009). Particu­ trate the representative morphological changes after LPS delivery. While larly in vitro, together with IFN-γ, it is canonically used to skew cells of sham and saline-injected areas show recognizable microglial macrophage lineage to a proinflammatory phenotype (Shapouri-Mog­ morphology, LPS-injected areas show rounded-shape cells, compatible haddam et al., 2018; Zhu et al., 2015). In vivo, the direct injection of LPS with vast monocyte infiltrate (Fig. 2E). Examination of sections, in fact, in the central nervous system (CNS) in rodents has commonly been used showed a massive population of OX42+ cells around vascular structures to study neuroinflammation, particularly as an inflammation-induced in the SNpc area (Supplementary Fig. S1), suggesting the contribution of PD model when injected directly into dopaminergic areas, specifically blood-infiltrated macrophages to the LAMM population at the injection the SNpc (Deng et al., 2020; Duty and Jenner, 2011). Inducing this site. proinflammatory phenotype in the CNS has been particularly useful to Importantly, since it is described that LAMM promote corralling in understand the harmful effects on neurons. Moreover, the hypothesis traumatic nervous system injuries (Zhou et al., 2020), we wanted to that bacterial-derived, LPS-mediated neuroinflammation could be analyze whether this phenomenon also occurred in the LPS-mediated behind PD pathogenesis makes this approach increasingly relevant neuroinflammatory scenario. For this, we use antibodies for glial (Brown, 2019). In fact, recent evidences point to unbalanced gut fibrillary acidic protein (GFAP) to detect reactive astrocytes in the microbiome as a potential contributor of the PD-related inflammation mesencephalon sections. Microscopy images revealed that GFAP (Romano et al., 2021), but how LPS or other membrane-bound bacterial immunoreactivity was higher in the LPS injected hemisphere (Fig. 3 A), components reach the brain is still to be determined (Wong, 2021). compared with the contralateral side. However, the core of the lesion It is clear that intranigral LPS injection generates reliable neuronal showed reduced GFAP reactivity concomitant to the accumulation of loss, which can be suitable for answering particular scientific questions LAMMs (Fig. 3A and B). Most interestingly, the border of the injury (Deng et al., 2020), but whether the inflammation of this model is displayed a dense population of GFAP+ astrocytes limiting the lesion, comparable to PD-like inflammation is still unclear. demonstrating the phenomenon of corralling in LPS induced dopami­ In the present work, we revisited the neuroinflammatory response nergic degeneration (Fig. 3C). generated by the direct injection of LPS in the SNpc in a rat model of A closer look at the GFAP anatomically enclosed, LPS-induced lesion dopaminergic degeneration and analyzed in detail with cellular reso­ site showed a densely packed LAMM area covering the entire injury lution the effects on the mesencephalic parenchyma. We observed that (Fig. 4A). Image analysis comparison of LPS-injured and intact SNpc, on the one hand, LPS-induced dopaminergic loss causes severe tissue using Voronoi partition or crystallization, showed a vast region of tiling damage characterized by microglial activation and massive macrophage at the lesion site (Fig. 4B) filling and compacting the injury. The 3D infiltration enclosed in an astrocyte-mediated corralled lesion showing blended view also showed a honeycomb-like tiling at the LPS-targeted high phagocytic capacity of dopaminergic debris, wound compaction parenchyma (Fig. 4C) similar to morphologies previously reported for and scar formation. These results indicate that although dopaminergic traumatic brain injury (Jassam et al., 2017). Cells were grouped loss is reproducible and reliable, the inflammation caused by the direct membrane-to-membrane along the SNpc most probably participating in injection of LPS does not recapitulate the typical inflammatory response the phagocytic clearance of debris. observed in ageing and PD. Because phagocytosis may be critical for injury healing of LPS- mediated dopaminergic elimination, we analyzed with high-resolution 2. Results detail the phagocytic capacity of LAMMs at the rats’ lesion sites. First, we identified a constellation pattern of TH+ material in the LPS lesion To revisit the effectiveness of LPS as a dopaminergic neurotoxin and area, with barely recognizable dopaminergic neuronal bodies (Fig. 5A). potential experimental model for PD, first, we analyzed the consistency Alpha blending and 3D reconstructions revealed a large amount of TH+ of a direct LPS injection inducing dopaminergic neuronal loss in the debris, within the volumetric space of the lesion, some of which was SNpc (Fig. 1A-C). We examined an early time window of three days to contained inside OX42+ LAMMs (Fig. 5B). Certain TH+ material was analyze the initial neuroinflammatory response and related neuro­ identifiable as neuronal bodies completely surrounded by LAMMs degeneration, and also at seven days to observe the potential recovery of (Fig. 5C and Supplementary video 1). High-resolution confocal Z scans the lesion. Precise intra-parenchymal LPS injection originated dopami­ confirmed that TH+ material was completely engulfed by LAMMs at the nergic depletion in the targeted site, observable both three and seven injection site, and 3D reconstructions facilitated the visualization of days after the injection, with no apparent effect on the contralateral side these phagocytic events (Fig. 5D, E, and Supplementary video 2). (Fig. 1D). Mainly dopaminergic neurons were eliminated but, interest­ Interestingly, the volume of TH+ debris, although reduced, persisted ingly, non-dopaminergic neurons, quantified as NeuN+ with no TH even seven days after the lesion (Supplementary Fig. S2) together with expression, also showed a similar percentage of reduction (Fig. 1 E-G) an important volumetric reduction of OX42+ membranes (Supplemen­ questioning the specificity and the particular vulnerability of dopami­ tary Fig. S2), indicating a slow and prolonged initiation of the wound nergic neurons to this endotoxin. At seven days, both dopaminergic and healing process. 2 P. Heman-Bozadas et al. Journal of Neuroimmunology 367 (2022) 577874 A C Sham Saline LPS TH 3 days B 50 Pm IPSILATERAL CONTRALATERAL TH 7 days 1 mm D IPSILATERAL CONTRALATERAL 25 E Sham LPS Number of TH+ neurons 20 TH NeuN DAPI 15 ** 3 days 10 5 0 25 20 Pm Number of TH+ neurons 20 15 * 7 days 10 G IPSILATERAL CONTRALATERAL Number of NeuN+/TH- neurons 5 25 0 20 15 * 3 days F Sham Saline LPS 10 5 TH NeuN DAPI 3 days 0 neurons 25 40 Pm 20 NeuN+/TH- 15 7 days & 7 days 10 Number of 5 0 Fig. 1. Intracranial injection of LPS induces lasting dopaminergic and non-dopaminergic neuronal loss. A. Diagram of the injection site in rat brain section. B. Photomosaic of a representative LPS-injected section at SNpc level with TH immunofluorescence. C. Representative confocal images of TH loss in the experimental groups. D. Quantification of dopaminergic neurons in ipsilateral and contralateral side, three and seven days after LPS injection. E. Representative high-resolution confocal images of immunofluorescence for TH (green) and NeuN (red) combined with DAPI (blue) counterstaining in intact and LPS injected SNpc (3 days after intracranial injection). F. Representative comparison of TH/NeuN in all experimental groups. G. Quantification of non-dopaminergic (NeuN+/TH− ) neurons in the SNpc, three and seven days after the injection. *p < 0.05, **p < 0.005 with respect to sham or &p < 0.05 to saline. Six animals were analyzed per group. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) 3 P. Heman-Bozadas et al. Journal of Neuroimmunology 367 (2022) 577874 Sham Saline LPS A C OX42 TH DAPI 3 days 50 Pm B OX42 DAPI IPSILATERAL CONTRALATERAL TH OX42 OX42 TH DAPI 7 days 1 mm OX42 DAPI TH OX42 DAPI D IPSILATERAL CONTRALATERAL 200 Number of OX42+ cells *& 150 3 days 100 50 Pm IPSILATERAL CONTRALATERAL 50 0 E 200 Number of OX42+ cells Sham Saline LPS 150 *& 7 days 100 * 50 0 Fig. 2. LAMMs massively populate LPS-induced dopaminergic lesion. A. Diagram of a representative rat brain section illustrating the location of the injection site applied to the SNpc. B. Photomosaic of section at the SNpc displaying the differences between the injected site (ipsilateral) and the non-injected site (contralateral) evidenced by immunofluorescence for TH and OX42. Representative confocal images from insets illustrating the contralateral and ipsilateral view in higher reso­ lution, evidenced by immunofluorescence for TH and OX42, and DAPI as a counterstaining are also shown. C. Representative images of OX42 expression in the SNpc of the three groups of animals (Sham, Saline and LPS), at the same anatomical level of the previous fig. D. Stereological quantification of OX42-expressing cells in the analyzed groups (3 days and 7 days after intracranial injections). *p < 0.001 with respect to sham; &p < 0.001 with respect to saline. E. Skeletonized diagrams of representative OX42+ cell morphology in the SNpc of the three experimental groups. Six animals were analyzed per group. In addition, although rare events, pyknotic nuclei could be seen in­ percentage of phagocytizing as on day three (Fig. 5G) evincing that side OX42+ cells, suggesting potential instances of primary phagocytosis elimination of dopaminergic cell fragments is a long-lasting process (Fig. 5F). Quantification of phagocytic events revealed high phagocytic during the course of the wound healing. activity at three days with a significant reduction of phagocytic events Because LPS injection caused a dramatic dopaminergic neuronal loss seven days after the lesion (Fig. 5G), indicating the evolving resolution accompanied by a massive LAMM corralling in tissue, we designed an in of the injury. However, persistent phagocytic activity of LAMMs after vitro platform to understand the initial roles of microglial-mediated LPS lesion was still detectable seven days after the injection with similar neuroinflammation in the primary stages of this scenario. First, to 4 P. Heman-Bozadas et al. Journal of Neuroimmunology 367 (2022) 577874 A IPSILATERAL CONTRALATERAL B IPSILATERAL CONTRALATERAL TH GFAP OX42 TH GFAP 1 mm 50 Pm C LESION BORDER GFAP DAPI GFAP TH DAPI OX42 TH DAPI MERGE 50 Pm Fig. 3. LPS-induced lesion induces GFAP astrocytic corraling in the SNpc. A. Composite mosaic of the mesencephalon of a representative LPS-injected rat shows GFAP overreactivity (red) corraling the lesion (limited by white arrows) at the ipsilateral hemisphere compared with contralateral side. Dopaminergic neurons (green) are also labeled with TH antibodies (representative image from 3 days after intracranial injection). B. Zoom-in micrography at the border of the lesion shows highly overreactive astrocytes limiting an area denselly populated by OX42+ cells (magenta). Contralateral side, at the same anatomical location, is also shown. C. Z confocal scan at the border reveals the high GFAP expression at the border compared with the site of the LPS lesion. DAPI+ nuclei (blue), TH+ dopaminergic neurons (green) and OX42+ LAMMs are also labeled (magenta). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) determine the direct toxicity of LPS, we treated dopaminergic cells with environment is also crucial for neuroinflammatory-mediated dopami­ increasing concentrations, and we observed only a slight dopaminergic nergic elimination (Fig. 7F-I). cell loss in some instances (Fig. 6A). Next, we separately treated microglia with the same protocol, detecting an increase in cell numbers, 3. Discussion but a relatively low or absent inflammatory response as determined by nitrite release and microglial cell size (Fig. 6B-D). In the present study, we show that LPS is an efficient compound Since a sole LPS administration does not explain the dramatic producing a lesion in the brain parenchyma that effectively depletes dopaminergic cell elimination, we co-cultured microglia and dopami­ mesencephalic neurons. Parallel to this lesion, a massive LAMM acti­ nergic cells to determine the role of the intercellular interactions. We vation occurs within the wounded area, populating the entire lesion observed a dramatic loss when combining both cell types (Fig. 6E-H), membrane-to-membrane and showing persistent phagocytosis of dopa­ with no apparent changes when adding increasing LPS concentrations, minergic debris. Importantly, the lesion shows the phenomenon of demonstrating that the presence of microglia appears to be essential for corralling, surrounded by a continuous palisade of reactive astrocytes. LPS-induced dopaminergic cell elimination (Fig. 6I and J) in the initial This wound compaction scenario juxtaposes the inflammatory land­ phases of pro-inflammatory injury. scape seen in ageing and PD (McGeer et al., 1988) or MPTP insult in Because this microglial-mediated elimination was spontaneous in humans and non-human primates (Barcia et al., 2004; Langston et al., this setting and apparently without an evident neuro-inflammatory 1999), where microglial cells appear activated within a preserved and stimulus, we wanted to confirm that the interaction with PC12 dopa­ recognizable nerve tissue structure. It is also different from the display of minergic cells in vitro does not trigger an inflammatory response. To do reactive astrocytes described in PD, which appear diffused throughout so, we analyzed microglial activation following PC12 presentation and the SNpc and without displaying corralling (McGeer and McGeer, 2008). compared it with the canonical proinflammatory stimulus LPS and IFN- In the case of rodent PD models, although a tangential infiltration of γ. The LPS/IFN-γ combination increased microglial activation parame­ monocytes/macrophages may be found, the anatomical architecture of ters in contrast with interaction solely with PC12, demonstrating that the parenchyma is not severely affected. MPTP insult in mice only causes the encounter with dopaminergic cells does not trigger this neuro- an early (24 h) and transient monocyte infiltration not altering the inflammatory response (Fig. 7A-E). outcome of dopaminergic degeneration (Parillaud et al., 2017), micro­ Knowing that the presence of microglia is critical for the elimination glia being the main cell phagocytizing dopaminergic neurons (Depboylu of dopaminergic cells, we postulated that phagocytosis might be a major et al., 2012). In 6-OHDA-injected rats, the situation is quite similar event in eliminating dopaminergic cell targets or debris. However, to (Akiyama and McGeer, 1989; Marinova-Mutafchieva et al., 2009), with understand the importance of the microglial-mediated toxic environ­ a preserved structure and limited monocyte infiltration (Espinosa-Oliva ment, we cultured dopaminergic cells with supernatant coming from et al., 2014). By contrast, the infiltration of monocytes/macrophages by naïve or activated microglia, and we observed a substantial reduction in intranigral injection of LPS appears to be relevant, and it has been dopaminergic cell numbers when adding supernatant from naïve and previously identified (Bok et al., 2018; Flores-Martinez et al., 2018; LPS/IFN-γ-activated microglia, demonstrating that the toxic Herrera et al., 2000), playing a central role in the LPS-dependent 5 P. Heman-Bozadas et al. Journal of Neuroimmunology 367 (2022) 577874 Sham LPS A OX42 TH DAPI OX42 TH DAPI 50 Pm B OX42 Image Voronoi Partition MC Crystallization PC Crystallization Sham LPS 50 Pm C OX42 DAPI OX42 DAPI 25 Pm Fig. 4. LAMM corralling generates honeycomb tiling at dopaminergic lesion site. A. Massive OX42+ (red) LAMM colonization of LPS-induced dopaminergic lesion in the SNpc (LPS) compared to an intact animal (Sham) (3 days after intracranial injection). Dopaminergic neurons are evidenced by TH (green) and nuclei (blue) are also counterstained (DAPI). B. Voronoi partition, monochrome (MC) and polychrome (PC) crystallization of OX42 images reveal dense tiling covering the injured areas in LPS-injected animals. C. LAMMs build a honeycomb-like tiling at lesion sites. Confocal images of OX42 cells (red) and DAPI+ nucleus (blue) are shown. Panel on the right illustrates the membrane-to-membrane apposition of OX42+ LAMMs at the SNpc. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) 6 P. Heman-Bozadas et al. Journal of Neuroimmunology 367 (2022) 577874 (caption on next page) 7 P. Heman-Bozadas et al. Journal of Neuroimmunology 367 (2022) 577874 Fig. 5. LAMMs exhibit persistent debris phagocytosis at LPS-injected site. A. Shadowed blend view of OX42/TH/DAPI confocal 3D capture at LPS-injected SNpc (3 days after intracranial injection). B. Top panel shows a 3D blend view at the LPS-injected SNpc. Bottom panel shows rotated higher magnification of tissue block across a central clipping plane displaying multiple phagocytic events (some examples are indicated with yellow arrowheads). C. On the left, high magnification confocal slide at lesion site showing dopaminergic neuronal debris. Central panel shows shadowed blend view evidencing dopaminergic debris inside and outside OX42 cells. Right panel shows the same image as a 3D rotation view at the lesion. D. High resolution confocal plane and lateral views at z axis, evidencing complete engulfing of TH debris by a OX42+ cell in the SNpc. E. Volumetric isosurface generation and rendering of the event showed in D, rotated and magnified on the right image. F. Confocal image of an OX42+ cell containing a pyknotic nucleus, evidenced by 5 Ramps scale. Measurement of relative fluorescence of both nuclei at the yellow line (Arrow indicates normal nucleus and arrowhead indicates pyknotic nucleus). G. Quantification of phagocytic events and percentage of OX42 phago­ cytizing at the LPS-lesioned SNpc. *p < 0.05. Six animals were analyzed per group. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) neuroinflammatory response in this anatomical area. Then, in subsequent phases, our results pinpoint phagocytosis as part Although previous reports highlight the utility of using the direct LPS of the wound compaction and healing of direct LPS-dependent lesion in injection to the rat’s SNpc (Herrera et al., 2000), because of the evident the CNS. Consistent with our in vivo images, this phagocytosis is evident inflammatory response, the parallelism with PD is still questionable. The and abundant three days after the lesion and, although reduced, remains direct contact of LPS with the nerve tissue generates an inflammatory- persistent at least seven days after the LPS insult. Besides, the fact that mediated damage that significantly deteriorates the parenchymal OX42+ cells completely surround dopaminergic neurons may also be structure. The resulting injury is so severe that some other structural interpreted as protective role in these phases of the injury resolution. proteins such as GFAP may not even be detected at the lesion core In this scenario, it seems that microglial-mediated neuro­ (Herrera et al., 2000). This is in fact compatible with our results in which inflammation, combining cytotoxicity and phagocytosis, is essential for GFAP reactivity is patent at the border of the injury but very limited at the dopaminergic elimination induced by LPS. However, the direct in­ the lesion core. Thus, a fair comparison between 6-OHDA and LPS in­ jection of LPS into the CNS generates such a massive wound, promoting jection is hard to convey. The only report comparing both models uses corralling, which significantly alters the structure of the tissue and puts an indirect approach, studying the LPS-induced neuroinflammation in in question the parallelism with the idiopathic parkinsonian inflamma­ the SNpc after a striatal injection (Parra et al., 2020). This is probably a tory response. This group of characteristics seems to be far from the PD- more adequate approach for a PD-like inflammatory reaction since like inflammation, rather being similar to other recently described sce­ injecting into the striatum avoids the direct damage caused by LPS in the narios for massive nerve tissue lesions, where clearance of debris, wound SNpc but generates an indirect microglial activation while structurally healing and compaction, mediated by LAMMs, contribute to the tissue preserving the area of interest (Deng et al., 2020). Besides, this approach repair (Zhou et al., 2020). may also better match the potential indirect access of LPS, or other In conclusion, our results demonstrate that direct LPS-mediated bacterial-derived components, to the brain parenchyma (Wong, 2021). lesion in the SNpc triggers a massive inflammatory response, charac­ Although in the present study we used a standardize LPS dose, we sug­ terized by LAMM tiling and phagocytosis that contributes to the wound gest that future studies may require adjusting the intracranial LPS compaction and healing. Although this model may be useful to generate dosage according to the manufacturers batch, to better establish the a drastic depletion of dopaminergic neurons, conclusions regarding the usefulness of this model for PD research. contribution of neuroinflammation in PD should be drawn cautiously. The phagocytic process observed is also very different between the Novel approaches and techniques need to be further explored and classical PD models and LPS. MPTP in mice induces a very specific re­ modeled to better understand the neuroinflammatory contribution to action against intoxicated dopaminergic neurons engulfing entire dopaminergic neurodegeneration in PD. neuronal bodies, enclosing pyknotic nuclei (Barcia et al., 2012). In contrast, in our images here, the phagocytosis of pyknotic nuclei is rare. 4. Materials and methods Rather, constellations of dopaminergic debris, which are not observable after MPTP, are being phagocytized by a large number of LAMMs in a 4.1. Animals and surgeries process of clearance. Although the damage generated by direct LPS injection is patent, the Thirty-six adult male Wistar rats (200–300 g, 2.5 months) were used mechanisms by which it occurs are to be further investigated. Parallel to for this study. Rats were housed in Plexiglas cages (33 × 44 × 20 cm) at traumatic brain injury, different phases may take place, including the the animal housing facilities (n = 5 per cage) under a 12/12 h light/dark initial phases, spanning minutes to hours, in which primary and sec­ cycle (light on at 08:00) at room temperature (RT) with water and food ondary damage occur followed by a subsequent phase of resolution, ad libitum. All experimental procedures were performed according to the covering several days (Jassam et al., 2017). Our co-cultures shed some International Guide for the Care and Use of Laboratory Animals (Na­ light on this phenomenon, at least in the initial phases of the damage, tional Research Council, 2011), register # ICS-2017-007 and the study indicating that sole delivery of LPS does not fully explain the dopami­ was approved by ethical committee of Instituto de Ciencias de la Salud nergic toxicity, but requires the interaction of microglia for effective from Universidad Veracruzana, and the NOM-062-ZOO-1999 and elimination. Probably, the presence of TLR4 receptors in myeloid cells NOM-087-ECOL-SSA1–2002 from Mexican legislation. All efforts were may in part clarify this outcome, microglia being the major TLR4- made to minimize animal discomfort during the study. Two sets of ex­ expressing cell in the nerve tissue in contrast with neurons (Lehnardt periments were planned according to the different time window be­ et al., 2002). This is consistent with the idea that a massive LPS delivery tween the intranigral injection of LPS and the euthanasia. In addition to within the CNS parenchyma triggers a LAMM-mediated inflammation a sham group, rats were injected either with an intracranial injection of that at first causes a large neuronal loss. This elimination may occur with LPS or saline in the SNpc. The groups of animals were then perfused the combination of cytotoxicity and phagocytosis, as supported by the in three (n = 18) or seven days (n = 18) after the LPS. vitro results. This indicates that, initially, the exacerbated response of the Three groups of animals (n = 6 per group) were used for each set of host is key to the elimination of neurons rather than the direct toxicity of experiments: group 1 (n = 6): sham (dura mater removed); group 2 (n = the endotoxin, consistent with previous reports (Brown, 2019; Bryant 6): saline (saline solution intranigral injection) and group 3 (n = 6): LPS et al., 2010). This is coherent with previous reports where the depletion (intranigral LPS injection). Animals were injected intranigrally with 10 of LAMMs by clodronate reduces the severity of LPS-induced brain μg/2 μL of LPS or saline solution at the same volume. In summary, 36 lesion in rodents (Lund et al., 2017; Zito et al., 2001), which appears rats were used for the histological assessment, 18 for each time point, particularly relevant in early time windows (Sheppard et al., 2019). distributed in groups of six animals. 8 P. Heman-Bozadas et al. Journal of Neuroimmunology 367 (2022) 577874 A 60 B 200 & 180 50 160 PC12 cell number BV2 cell number 40 140 120 * * 30 ** 100 80 20 60 10 40 20 0 0 PC12 PC12 PC12 PC12 PC12 PC12 PC12 PC12 PC12 BV2 BV2 BV2 BV2 BV2 BV2 BV2 BV2 BV2 CTRL LPS LPS LPS LPS LPS LPS LPS LPS CTRL LPS LPS LPS LPS LPS LPS LPS LPS 50 100 250 500 1000 1500 2000 2500 50 100 250 500 1000 1500 2000 2500 C 7 D 350 CD11b area per BV2 cell (Pm2) NO2- mM equivalent for BV2 6 300 5 250 4 200 3 150 2 100 1 50 0 0 BV2 BV2 BV2 BV2 BV2 BV2 BV2 BV2 BV2 BV2 BV2 BV2 BV2 BV2 BV2 BV2 BV2 BV2 CTRL LPS LPS LPS LPS LPS LPS LPS LPS CTRL LPS LPS LPS LPS LPS LPS LPS LPS 50 100 250 500 1000 1500 2000 2500 50 100 250 500 1000 1500 2000 2500 E PC12 BV2 and PC12 F 60 TH DAPI CD11b TH DAPI 50 PC12 cell number 40 30 20 * 10 50 m 0 PC12 PC12 and BV2 PC12 P. Microglia and PC12 G H 60 TH DAPI OX42 TH DAPI 50 PC12 cell number 50 40 30 20 $ 15 10 50 m 5 0 PC12 PC12 and P. Microglia I 40 J 1.2 35 * 1.0 PC12 cell number/field NO2- PM equivalents 30 0.8 25 20 0.6 15 & 0.4 10 0.2 5 0 0 PC12 PC12 PC12 PC12 BV2 BV2 BV2 BV2 PC12 BV2 BV2 BV2 BV2 BV2 alone LPS LPS LPS PC12 PC12 PC12 PC12 alone alone PC12 PC12 PC12 PC12 50 500 1500 LPS LPS LPS LPS LPS LPS 50 500 1500 50 500 1500 (caption on next page) 9 P. Heman-Bozadas et al. Journal of Neuroimmunology 367 (2022) 577874 Fig. 6. LPS by itself is slightly toxic to PC12 dopaminergic cells and scarcely increases microglial-mediated neuro-inflammatory response while the presence of microglia spontaneously eliminates dopaminergic cells. A. Quantification of PC12 dopaminergic cell density after LPS treatment in cell culture. *p < 0.01 and **p < 0.001 with respect to CTRL. B. Quantification of microglial cell number after increasing concentrations of LPS. &p < 0.001 with respect to the other conditions. C. Concentration of nitrites released by microglia after LPS treatment. No differences were seen with respect to CTRL. D. Quantification of CD11b expressing area in microglial cells after LPS treatment. No differences were seen with respect to the CTRL. E. Representative micrographies of dopaminergic cell culture, labeled with TH antibodies (green), before and after adding BV2 microglial cells, labeled with CD11b antibodies (red). DAPI was used as counterstaining. F. Quantification of PC12 dopaminergic cells before and after adding BV2 microglia. *p < 0.05 with respect to PC12 alone. G. Representative pictures of TH+ dopaminergic cells (green), before and after adding primary microglia, labeled with OX42 antibodies (red) in combination with DAPI (blue). H. Quantification of TH+ cells before and after co- culture with primary microglia. $p < 001, compared with PC12 alone. I. Quantification of dopaminergic cells after microglial presentation with increasing con­ centration of LPS. *p < 0.01 with respect to PC12 alone. &p < 0.001 with respect to conditions without microglia. J. Quantification of nitrites release after LPS treatment (Analyses and quantifications were made after 24 h of treatment for each condition). No differences were seen between treatments. Each experiment had three replicates. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) Intracranial surgery was done as follows. Rats were anesthetized done with TBS-Triton 0.05% and tissues were incubated with fluores­ with a single intraperitoneal dose of ketamine/xylazine (10 mg/kg/8 cent secondary antibodies: Alexa Fluor 488 (goat anti-rabbit IgG) mg/kg) and received an intranigral injection of LPS from Escherichia Coli (Thermofisher, Waltham, MA, USA), Alexa Fluor 488 (goat anti-mouse 055: B5) (Sigma-Aldrich; St. Louis, MO, USA) or saline. We used the IgG) (Thermofisher, Waltham, MA, USA) Alexa Fluor 555 (Goat anti following coordinates: AP +3.0 mm, from the interaural point; ML +2.8 Chicken Ig) (Thermofisher, Waltham, MA, USA), and Alexa Fluor 555 mm from the intraparietal suture and DV -6.9 mm from the dura mater. goat anti-mouse IgG (Thermofisher, Waltham, MA, USA), for four hours The injection flow was 0.2 μL/min maintained by a perfusion micro­ (at a concentration of 1:1000 in horse serum 1% + sodium azide 0.1%) pump (Stoelting, Wood Dale, IL, USA) according to previous procedure and protected from light. Next, three washes were done with PBS for 10 (Hernandez-Baltazar et al., 2013). Once the LPS or saline solution was min and tissues were incubated with DAPI (1:1000) (Thermofisher, injected, according to each case, the needle was left inside five minutes Waltham, MA, USA), for 30 min to label the cell nuclei. They were rinsed to allow complete diffusion, then was withdrawn slowly after which the with PBS three times for 10 min and finally were mounted on non- animals were sutured. gelatinized slides and protected with antifading reagent (Pro-Long Gold, Thermofisher, Waltham, MA, USA) and coverslips. 4.1.1. Perfusion Three or seven days after surgery, animals were deeply anesthetized 4.2. Image capture and quantifications with 100 mg/kg i.p. of sodium pentobarbital (SedalPharma®, Labo­ ratorios Pet’s Pharma México), and perfused transcardially with 200 mL TH and Iba-1 immunoreactivity were visualized under the multi­ of 0.1 M PBS (from 1 M PBS, 8.1 mM Na2HPO4, 1.2 mM KH2PO4, 138 channel epifluorescence microscope (Eclipse 90i, Nikon), adapted to a mM NaCl, 2.7 mM of KCl, filtered, pH 7.4) and with 100 mL of 4% high-resolution camera (Nikon DXM1200F), using software to system­ paraformaldehyde in 0.1 M PBS using a peristaltic pump. During the atically capture images (ACT-1). Representative mosaic images of the perfusion, only the head of the animal was favored, so it was necessary SNpc were also taken with a high-resolution camera with a microscope to mechanically occlude the descending aortic artery. with an automated stage (Eclipse TE2000-E, Nikon) covering the section with systematic images according to x and y coordinates. Image mosaics 4.1.2. Extraction of brain tissue were built using a stitching tool plugin (Image J). The number of cells Once the perfusion was completed, the brains were immediately was quantified using a specialized software (Image J) and considering removed and post-fixed for 24 h at 4 ◦ C in 4% paraformaldehyde. After the physical dissector as stereological criteria. Three representative this, they were placed in 30% sucrose solution in 0.1 M PBS under the sections of the mesencephalon containing the SNpc were imaged, and same storage conditions. Subsequently, brains were cut in 35 μm-thick nine sampling dissectors covering the SNpc were quantified per hemi­ serial sections in a cryostat (Leica CM1520, Leica Inc., Germany) in sphere. Each dissector was quantified with the inclusion and exclusion coronal plane to obtain a representative sample of all levels of the SNpc. criterion according to previous protocols (Gundersen, 1986). The 35 μm slices were collected starting from the anterior-most portion of the SNpc and sequentially placed with a soft-bristled brush inside a 4.3. Confocal microscopy sterile 24-well box (Nalgene), previously filled with 0.1 M PBS. Sections were placed one by one in six series; this allowed 9–10 representative TH/OX42 and TH/NeuN positive cells were visualized using a high- sections of SNpc to be obtained for each well. The tissue was stored in resolution confocal microscope (Zeiss LSM 700) with a 20× objective cryoprotective solution at − 20 ◦ C until used. and capturing systematic images of the SNpc with specialized software (ZEN 2010, Zeiss). For quantifications, TH+/OX42+ or TH+/NeuN+ 4.1.3. Immunofluorescence immunofluorescence images were captured in 3D stacks configured with Tissues were washed three times for 10 min with PBS containing several optical sections with a 1-μm optical interval and analyzed with 0.13 M NaCl, 0.010 M Na2HPO4 and 0.002 M NaH2PO4 at pH 7.4. Next, specialized software. In addition, DAPI was also captured as counter­ they were immersed in citrate buffer at 60–80 ◦ C for 20 min and rinsed staining. For numerical quantifications, including number of cells and again with PBS for five minutes and after with TBS-Triton 0.05%. Sub­ the number phagocytic events, we used object counter tools (Image J) sequently, they were incubated for one hour in 10% horse serum +0.1% and optical dissector stereological criteria. To quantify volumes and sodium azide for blocking nonspecific sites, then washed five minutes visualize phagocytosis events, we used different three-dimensional with horse serum 1% + sodium azide 0.1% and incubated with primary rendering software (Imaris, Bitplane and Illucida FX, Los Angeles) to antibodies [anti-TH (rabbit, 1:1000) (Merck-Millipore, Darmstadt, create isosurfaces and blend views. To visualize Voronoi partition and Germany) for identification of dopaminergic neurons; anti-OX42 crystallization, we also used specialized software applications (ImageJ (mouse, 1:500) (Abcam, Cambridge, United Kingdom) and Iba-1 (rab­ and Pixelmator). Analysis and visualization of relative florescence with bit, 1:500) (Wako Chemicals, Hong Kong) for microglial cells; anti-NeuN 5 ramps was also done with specific application software (Image J). (mouse, 1:300) (Merck Millipore, Darmstadt, Germany), to detect neuronal nuclei, and anti-GFAP (Chicken, 1:500) (Abcam, Cambridge, United Kingdom)] in 1% horse serum solution +0.1% sodium azide for 48 h at room temperature (RT). After that, three 10-min washes were 10 P. Heman-Bozadas et al. Journal of Neuroimmunology 367 (2022) 577874 Fig. 7. Cytotoxicity from activated microglia is deleterious for dopami­ nergic cells. A. Presentation of PC12 dopaminergic cells does not activate BV2 microglia. Representative microg­ raphies of cultured BV2 microglia after LPS with increasing concentrations of IFN-γ, or addition of PC12. B. Quanti­ fication of CD11b expressing area in BV2 microglia after treatments or PC12 presentation. &p < 0.001 with the rest of the conditions. C. Quantification of nitrites released by microglia after experimental conditions. *p < 0.05 and **p < 0.001 with respect to BV alone, &p <0.001 with respect to PC12 with BV2. D. Representative pictures of mesencephalic primary microglia after LPS with increasing concentrations of IFN-γ, and co-cultures with dopami­ nergic cells PC12. E. Quantification of OX42 expresing area in primary microglia after treatments and inter­ action with PC12. $p < 0.01 &p < 0.001 with respect to Microglia-PC12. F. Diagram of the experimental design. G. Representative microscopy images of PC12 dopaminergic cells alone and after supernatant treatment from LPS/IFN-γ-treated microglia. H. Measurements of nitrites released in the experimental conditions (top chart), and quantification of dopami­ nergic cells after treatments. &p < 0.05 with respect to PC12 alone and sup from naïve. *p < 0.05 with respect to PC12 alone. **p < 0.001 with respect to PC12 alone. (Analyses and quantifi­ cations were made after 24 h of treat­ ment for each condition). Each experiment had three replicates. I. Quantification of dopaminergic cells after treatment with media (either suited for PC12 or for primary micro­ glia), supernatant from naïve or LPS/ IFN-γ-treated primary microglia. *p < 0.05, ****p < 0.001 with respect to PC12 medium. ###p < 0.01, ####p < 0.001 with respect to primary medium. 11 P. Heman-Bozadas et al. Journal of Neuroimmunology 367 (2022) 577874 5. Cell culture experiments mentioned for BV2-PC12 co-cultures, after 24 h cells were fixed with 4% paraformaldehyde in PBS for immunocytofluorescence. Analogously to 5.1. Cell lines BV2 cells, supernatant from naïve or LPS/IFN-γ-activated mesencephalic primary microglia was also used to treat PC12 cells. PC12 and BV2 cells were obtained from the INc-UAB institutional repository. BV2 is an immortalized murine microglia cell line, which has 5.3. Immunocytofluorescence been widely used and successfully evaluated in response to LPS and IFN- γ (Henn et al., 2009). Both cell lines were maintained at 37 ◦ C with 5% The immunocytofluorescence was performed on cell cultures to CO2 in their corresponding media, which for the PC12 cell line was simultaneously detect TH and CD11b (in BV2) or OX42 (in primary Dulbecco’s Modified Eagle Medium (DMEM) (Ref#, 41,965,039, Ther­ microglia). Cells were washed with PBS, then permeabilized with 0.02% mofisher, Waltham, MA, USA) supplemented with 7% fetal bovine saponin/PBS at RT and later rewashed with PBS1×. Nonspecific sites serum, 7% horse serum and 0.2% penicillin-streptomycin and for the were first blocked with PBS1×, 0.01% saponin and 0.075% glycine and BV2 cell line was Roswell Park Memorial Institute medium (RPMI) (Ref later nonspecific sites were again blocked with PBS1×, 0.01% saponin, #, 21,875–034, Thermofisher, Waltham, MA, USA) supplemented with 0.075% glycine and 5% BSA. Coverslips containing the cells were placed 10% fetal bovine serum and 0.1% penicillin- streptomycin. For the in a humid chamber with 12 μL of PBS1×, 0.01% saponin, 1% BSA and different experiments, PC12 and/or BV2 were placed in a 24-well cul­ the primary antibody overnight. To detect TH and CD11b, the primary ture plate and grown for 24 h then treated with different compounds antibodies were anti-TH (sheep, 1:500) (Merck; Darmstadt, Germany), such as LPS at different concentrations (from 50 ng/mL to 2500 ng/mL anti-CD11b (rat, 1:1000) (Abcam; Cambridge, United Kingdom) and and 10 ng/mL when combined with IFN-γ), IFN-γ (from 10 ng/mL to 50 anti-OX42 (mouse, 1:500) (Abcam; Cambridge, United Kingdom), ng/mL), or co-cultured with PC12. The process to carrying out the BV2 respectively. The following day coverslips were placed again in the 24- microglial and PC12 dopaminergic cells co-culture started with plating well plate, cells were washed with PBS 1X before being placed again in a BV2 in poly-L-lysine-coated coverslips in a 24-well culture plate in a humid chamber with PBS1X, 0.01% saponin, 1% BSA and the secondary concentration of 50.000cells/mL with 500 μL of DMEM medium. They antibody for 45 min. In order to detect TH, the secondary antibody was were allowed to grow in an incubator at 36 ◦ C and 5% of CO2 for 24 h. donkey anti-sheep (1:1000) in green fluorescence (Alexa Fluor 488) BV2/PC12 co-culture was performed by the addition of PC12 cultured (Life Technologies; Carlsbad, CA, USA), for CD11b detection the sec­ cells (500 μL) in a concentration of 100.000 cells/mL in the wells pre­ ondary antibody was goat anti-rat (1:500) in red fluorescence (Alexa viously containing BV2 cell culture. Twenty-four hours after the treat­ Fluor 555) (Bio-Rad Company; Berkeley, California, USA) and for OX42 ment, the supernatant was collected to conduct the Griess assay or to goat anti-mouse (1:100) also in red fluorescence (Alexa Fluor 555) (Life treat subsequent cultures. At the end of the experiments, cells were fixed Technologies; Carlsbad, CA, USA). Coverslips were later returned to the with 4% paraformaldehyde in PBS for immunocytofluorescence. 24-well plate to be washed with PBS1X before adding 400 μL per well of the nucleus marker, DAPI (1:1000) (Life technologies; Carlsbad, CA, 5.2. Primary cultures of midbrain microglia USA) in PBS1X. One more wash with PBS 1X was done before the cov­ erslips were mounted on a glass slides using antifade reagent (Prolong Primary microglia was obtained from rats P0-P2 (OFA, Sprague Gold, Life Technologies; Carlsbad, CA, USA). For every experimental Dawley). Rat pups were euthanized, and the midbrain was dissected and condition, a secondary antibody control was also performed, leaving one placed in a petri dish with PBS 1× supplemented with glucose. After this, coverslip per condition without any primary antibodies. a set of fresh solutions was prepared to get mixed glia from the midbrain. Solution 1 containing 50 mL Krebs 1×, 0.15 g BSA and 0.4 mL of Mg2+, 5.4. Immunocytofluorescence quantification solution 2 combining 10 mL of solution 1 and 2.5 mg of trypsin, solution 3 prepared with 10 mL from solution 1, 0.8 mg DNase, 5.2 mg trypsine To quantify the number of cells in the co-culture and its controls after inhibitor and 0.1 mL Mg2+, solution 4 prepared with 8.4 mL from so­ treatments, we used an image analysis protocol. Each coverslip was lution 1 and 1.6 mL of solution 3, and, finally solution 5 containing 5 mL imaged using a fluorescence microscope (Nikon Eclipse 90i) attached to of solution 1, 40 μL of Mg2+ and 6 μL Ca2+. Then, all collected midbrains a DXM 1200F digital camera and version 2.70 of the ACT-1 software were placed in solution 1 and centrifuged for 30 s at 1500 rpm, followed (Nikon Corporation). With this system, we obtained 30 images per by suctioning the supernatant and adding solution 2 to the pellet, next, condition at 20×. Two quantification methods were used for the this was incubated at 37 ◦ C for 10 min and later the solution 4 was added immunocytofluorescence labeling. One consisted of counting the num­ to the pellet together with solution 2, in order to proceed to the enzy­ ber of cells, either PC12 or BV2, following stereological criteria and matic digestion, afterwards tubes were spin at 1500 rpm. The superna­ using a specialized software (Image J version 1.47, NIH, USA). The other tant was discarded, and 2–3 mL of solution 3 were added, then the cells method consisted of measuring the area of BV2 (CD11b) to estimate the were passed through a mesh to separate them. Finally, the rest of solu­ activation, also using the same software (Image J version 1.47, NIH, tion 3 was added through the mesh and all the content was added to USA). solution 5, which was centrifuged at 1000 rpm for 5 min. After this, the supernatant was discarded and the pellet was re-suspended in 5 mL of 5.5. Determination of nitrites by Griess assay mixed glia culture medium (DMEM medium, 10% fetal bovine serum and 1% penicillin-streptomycin). From this solution, 10 μL were taken The determination of nitrites by Griess assay is based on a chemical and placed in an Eppendorf tube with 10 μL of tripan blue, and from the reaction in which nitric oxide in the presence of an aromatic amine latter solution, 10 μL were taken and placed in a neubauer chamber to produces different compounds in sequence, resulting in a pink-colored estimate the cell density. Cells were counted in order to add to the flask compound, easily detectable by spectrophotometry. First, the calibra­ in a known concentration of mixed glia, in our case, 50,000 cells/mL. tion curve was prepared with known concentrations of NaNO2 (100 μM, The rest of the volume of the flask was filled up with mixed glia culture 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.125 μM, 1.5625 μM and 0.78125 medium. Once the mixed glia grew, the flask containing this glia was μM), and 100 μL of each solution was added into a 96-well plate for the shaken at 300 rpm during 3 h, then the supernatant was collected to get calibration curve, as was 100 μL of the experiment samples. Next, 100 μL the primary microglia since they grow in the superficial layers of the of the Griess reagent (0.1 g in 2.5 mL milli-Q water) was added to every culture, consecutively primary microglia were placed in 24-well culture well. Duplicates were done for each condition. The plate was then plate, letting the cells to grow up to 24 h. Then, they were treated with incubated in darkness at RT for 15 min. 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