Publications by authors named "Sujatha Kannan"

92 Publications

Microglial Metabolism After Pediatric Traumatic Brain Injury - Overlooked Bystanders or Active Participants?

Front Neurol 2020 25;11:626999. Epub 2021 Jan 25.

Department of Anesthesiology and Critical Care Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, United States.

Microglia play an integral role in brain development but are also crucial for repair and recovery after traumatic brain injury (TBI). TBI induces an intense innate immune response in the immature, developing brain that is associated with acute and chronic changes in microglial function. These changes contribute to long-lasting consequences on development, neurologic function, and behavior. Although alterations in glucose metabolism are well-described after TBI, the bulk of the data is focused on metabolic alterations in astrocytes and neurons. To date, the interplay between alterations in intracellular metabolic pathways in microglia and the innate immune response in the brain following an injury is not well-studied. In this review, we broadly discuss the microglial responses after TBI. In addition, we highlight reported metabolic alterations in microglia and macrophages, and provide perspective on how changes in glucose, fatty acid, and amino acid metabolism can influence and modulate the microglial phenotype and response to injury.
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http://dx.doi.org/10.3389/fneur.2020.626999DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7868439PMC
January 2021

Dendrimer-tesaglitazar conjugate induces a phenotype shift of microglia and enhances β-amyloid phagocytosis.

Nanoscale 2021 Jan;13(2):939-952

Center for Nanomedicine, Department of Ophthalmology, Wilmer Eye Institute Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA. and Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD 21218, USA and Hugo W. Moser Research Institute at Kennedy Krieger, Inc., Baltimore, MD 21205, USA.

Switching microglia from a disease exacerbating, 'pro-inflammatory' state into a neuroprotective, 'anti-inflammatory' phenotype is a promising strategy for addressing multiple neurodegenerative diseases. Pro-inflammatory microglia contribute to disease progression by releasing neurotoxic substances and accelerating pathogenic protein accumulation. PPARα and PPARγ agonists have both been shown to shift microglia from a pro-inflammatory ('M1-like') to an alternatively activated ('M2-like') phenotype. Such strategies have been explored in clinical trials for neurological diseases, such as Alzheimer's and Parkinson's disease, but have likely failed due to their poor blood-brain barrier (BBB) penetration. Hydroxyl-terminated polyamidoamine dendrimers (without the attachment of any targeting ligands) have been shown to cross the impaired BBB at the site of neuroinflammation and accumulate in activated microglia. Therefore, dendrimer conjugation of a PPARα/γ dual agonist may enable targeted phenotype switching of activated microglia. Here we present the synthesis and characterization of a novel dendrimer-PPARα/γ dual agonist conjugate (D-tesaglitazar). In vitro, D-tesaglitazar induces an 'M1 to M2' phenotype shift, decreases secretion of reactive oxygen species, increases expression of genes for phagocytosis and enzymatic degradation of pathogenic proteins (e.g. β-amyloid, α-synuclein), and increases β-amyloid phagocytosis. These results support further development of D-tesaglitazar towards translation for multiple neurodegenerative diseases, especially Alzheimer's and Parkinson's Disease.
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http://dx.doi.org/10.1039/d0nr05958gDOI Listing
January 2021

NMDA Receptor Antagonism for Neuroprotection in a Canine Model of Hypothermic Circulatory Arrest.

J Surg Res 2020 Dec 18;260:177-189. Epub 2020 Dec 18.

Division of Cardiac Surgery, Department of Surgery, Johns Hopkins University School of Medicine, Baltimore, Maryland. Electronic address:

Background: Hypothermic circulatory arrest (HCA) is associated with neurologic morbidity, in part mediated by activation of the N-methyl-D-aspartate glutamate receptor causing excitotoxicity and neuronal apoptosis. Using a canine model, we hypothesized that the N-methyl-D-aspartate receptor antagonist MK801 would provide neuroprotection and that MK801 conjugation to dendrimer nanoparticles would improve efficacy.

Materials And Methods: Male hound dogs were placed on cardiopulmonary bypass, cooled to 18°C, and underwent 90 min of HCA. Dendrimer conjugates (d-MK801) were prepared by covalently linking dendrimer surface OH groups to MK801. Six experimental groups received either saline (control), medium- (0.15 mg/kg) or high-dose (1.56 mg/kg) MK801, or low- (0.05 mg/kg), medium-, or high-dose d-MK801. At 24, 48, and 72 h after HCA, animals were scored by a standardized neurobehavioral paradigm (higher scores indicate increasing deficits). Cerebrospinal fluid was obtained at baseline, eight, 24, 48, and 72 h after HCA. At 72 h, brains were examined for histopathologic injury in a blinded manner (higher scores indicate more injury).

Results: Neurobehavioral deficit scores were reduced by low-dose d-MK801 on postoperative day two (P < 0.05) and by medium-dose d-MK801 on postoperative day 3 (P = 0.05) compared with saline controls, but free drug had no effect. In contrast, high-dose free MK801 significantly improved histopathology scores compared with saline (P < 0.05) and altered biomarkers of injury in cerebrospinal fluid, with a significant reduction in phosphorylated neurofilament-H for high-dose MK801 versus saline (P < 0.05).

Conclusions: Treatment with MK-801 demonstrated significant improvement in neurobehavioral and histopathology scores after HCA, although not consistently across doses and conjugates.
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http://dx.doi.org/10.1016/j.jss.2020.11.075DOI Listing
December 2020

Dendrimer-Mediated Targeted Delivery of Rapamycin to Tumor-Associated Macrophages Improves Systemic Treatment of Glioblastoma.

Biomacromolecules 2020 12 28;21(12):5148-5161. Epub 2020 Oct 28.

Center for Nanomedicine, Department of Ophthalmology, Wilmer Eye Institute Johns Hopkins University School of Medicine, Baltimore, Maryland 21231, United States.

Glioblastoma exhibits high mortality rates due to challenges with drug delivery to the brain and into solid tumors. This two-pronged barrier necessitates high doses of systemic therapies, resulting in significant off-target toxicities. Recently, dendrimer-nanomedicines (without ligands) have shown promise for targeting specific cells in brain tumors from systemic circulation, for improved efficacy and amelioration of systemic toxicities. A dendrimer-rapamycin conjugate (D-Rapa) is presented here that specifically targets tumor-associated macrophages (TAMs) in glioblastoma from systemic administration. D-Rapa improves suppression of pro-tumor expression in activated TAMs and antiproliferative properties of rapamycin in glioma cells . , D-Rapa localizes specifically within TAMs, acting as depots to release rapamycin into the tumor microenvironment. This targeted delivery strategy yields improved reduction in tumor burden and systemic toxicities in a challenging, clinically relevant orthotopic syngeneic model of glioblastoma, demonstrating the significant potential of dendrimers as targeted immunotherapies for improving glioblastoma treatment, still an unmet need.
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http://dx.doi.org/10.1021/acs.biomac.0c01270DOI Listing
December 2020

Systemic dendrimer-drug nanomedicines for long-term treatment of mild-moderate cerebral palsy in a rabbit model.

J Neuroinflammation 2020 Oct 25;17(1):319. Epub 2020 Oct 25.

Department of Anesthesiology and Critical Care, Johns Hopkins School of Medicine, Baltimore, MD, USA.

Background: Neuroinflammation mediated by microglia plays a central role in the pathogenesis of perinatal/neonatal brain injury, including cerebral palsy (CP). Therapeutics mitigating neuroinflammation potentially provide an effective strategy to slow the disease progression and rescue normal brain development. Building on our prior results which showed that a generation-4 hydroxyl poly(amidoamine) (PAMAM) dendrimer could deliver drugs specifically to activated glia from systemic circulation, we evaluated the sustained efficacy of a generation-6 (G6) hydroxyl-terminated PAMAM dendrimer that showed a longer blood circulation time and increased brain accumulation. N-acetyl-L-cysteine (NAC), an antioxidant and anti-inflammatory agent that has high plasma protein binding properties and poor brain penetration, was conjugated to G6-PAMAM dendrimer-NAC (G6D-NAC). The efficacy of microglia-targeted G6D-NAC conjugate was evaluated in a clinically relevant rabbit model of CP, with a mild/moderate CP phenotype to provide a longer survival of untreated CP kits, enabling the assessment of sustained efficacy over 15 days of life.

Methods: G6D-NAC was conjugated and characterized. Cytotoxicity and anti-inflammatory assays were performed in BV-2 microglial cells. The efficacy of G6D-NAC was evaluated in a rabbit model of CP. CP kits were randomly divided into 5 groups on postnatal day 1 (PND1) and received an intravenous injection of a single dose of PBS, or G6D-NAC (2 or 5 mg/kg), or NAC (2 or 5 mg/kg). Neurobehavioral tests, microglia morphology, and neuroinflammation were evaluated at postnatal day 5 (PND5) and day 15 (PND15).

Results: A single dose of systemic 'long circulating' G6D-NAC showed a significant penetration across the impaired blood-brain-barrier (BBB), delivered NAC specifically to activated microglia, and significantly reduced microglia-mediated neuroinflammation in both the cortex and cerebellum white matter areas. Moreover, G6D-NAC treatment significantly improved neonatal rabbit survival rate and rescued motor function to nearly healthy control levels at least up to 15 days after birth (PND15), while CP kits treated with free NAC died before PND9.

Conclusions: Targeted delivery of therapeutics to activated microglia in neonatal brain injury can ameliorate pro-inflammatory microglial responses to injury, promote survival rate, and improve neurological outcomes that can be sustained for a long period. Appropriate manipulation of activated microglia enabled by G6D-NAC can impact the injury significantly beyond inflammation.
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http://dx.doi.org/10.1186/s12974-020-01984-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7586697PMC
October 2020

Targeting Mitochondria in Tumor-Associated Macrophages using a Dendrimer-Conjugated TSPO Ligand that Stimulates Antitumor Signaling in Glioblastoma.

Biomacromolecules 2020 09 31;21(9):3909-3922. Epub 2020 Aug 31.

Mitochondria mediate critical cellular processes, including proliferation, apoptosis, and immune responses; as such, their dysfunction is pathogenic in many neurodegenerative disorders and cancers. In glioblastoma, targeted delivery of mitochondria-focused anticancer therapies has failed to translate into clinical success due to the nonspecific cellular localization, heterogeneity of receptor expression across patients, poor transport across biological barriers to reach the brain, tumor, and mitochondria, and systemic side effects. Strategies that can overcome brain and solid tumor barriers and selectively target mitochondria within specific cell types may lead to improvements in glioblastoma treatment. Developments in dendrimer-mediated nanomedicines have shown promise targeting tumor-associated macrophages (TAMs) in glioblastoma, following systemic administration. Here, we present a novel dendrimer conjugated to the translocator protein (18 kDa) (TSPO) ligand 5,7-dimethylpyrazolo[1,5-α]pyrimidin-3-ylacetamide (DPA). We developed a clickable DPA for conjugation on the dendrimer surface and demonstrated that the dendrimer-DPA conjugate (D-DPA) significantly increases dendrimer colocalization with mitochondria. Compared to free TSPO ligand PK11195, D-DPA stimulates greater antitumor immune signaling. , we show that D-DPA targets mitochondria specifically within TAMs following systemic administration. Our results demonstrate that dendrimers can achieve TAM-specific targeting in glioblastoma and can be further modified to target specific intracellular compartments for organelle-specific drug delivery.
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http://dx.doi.org/10.1021/acs.biomac.0c01033DOI Listing
September 2020

Glial restricted precursor delivery of dendrimer N-acetylcysteine promotes migration and differentiation following transplant in mouse white matter injury model.

Nanoscale 2020 Aug 29;12(30):16063-16068. Epub 2020 Jul 29.

Moser Center for Leukodystrophies, Kennedy Krieger Institute, Baltimore, MD, USA.

Oligodendrocyte replacement using glial restricted precursors (GRPs) is a promising avenue for the treatment of acquired or genetic white matter disorders; however, limited long-term survival of these cells post-transplant may impede maximal recovery. Nanotherapeutic approaches can facilitate stem cell delivery while simultaneously delivering factors aimed at enhancing and nourishing stem cells en route to, and at, the target site. Hydroxyl polyamidoamine (PAMAM) dendrimer nanoparticles have been used in a variety of models to deliver therapeutics in a targeted manner to injury sites at low doses. Here, survival and migration of GRPs was assessed in a mouse model of neonatal white matter injury with different methods of dendrimer nanoparticle support. Our findings demonstrate the ability of GRPs to take up nanoparticle-drug conjugates and for these conjugates to act beyond the injury site in vivo. Compared to GRPs alone, mice receiving dendrimer-drug in parallel to GRPs, or via GRPs as the delivery vector, showed improved migration and differentiation of cells 8 weeks post-transplant. These studies demonstrate that drug-conjugated nanoparticles can enhance transplanted progenitor cell survival and migration, and suggest that combination therapies may allow engraftment without overt immunosuppression.
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http://dx.doi.org/10.1039/c9nr10804aDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7448752PMC
August 2020

Glutamine Antagonist JHU-083 Normalizes Aberrant Hippocampal Glutaminase Activity and Improves Cognition in APOE4 Mice.

J Alzheimers Dis 2020 ;77(1):437-447

Departments of Psychiatry and Behavioral Sciences, Johns Hopkins University, Baltimore, MD, USA.

Background: Given the emergent aging population, the identification of effective treatments for Alzheimer's disease (AD) is critical.

Objective: We investigated the therapeutic efficacy of JHU-083, a brain-penetrable glutamine antagonist, in treating AD using the humanized APOE4 knock-in mouse model.

Methods: Cell culture studies were performed using BV2 cells and primary microglia isolated from hippocampi of adult APOE4 knock-in mice to evaluate the effect of JHU-083 treatment on LPS-induced glutaminase (GLS) activity and inflammatory markers. Six-month-old APOE4 knock-in mice were administered JHU-083 or vehicle via oral gavage 3x/week for 4-5 months and cognitive performance was assessed using the Barnes maze. Target engagement in the brain was confirmed using a radiolabeled GLS enzymatic activity assay, and electrophysiology, gastrointestinal histology, blood chemistry, and CBC analyses were conducted to evaluate the tolerability of JHU-083.

Results: JHU-083 inhibited the LPS-mediated increases in GLS activity, nitic oxide release, and pro-inflammatory cytokine production in cultured BV2 cells and primary microglia isolated from APOE4 knock-in AD mice. Chronic treatment with JHU-083 in APOE4 mice improved hippocampal-dependent Barnes maze performance. Consistent with the cell culture findings,postmortem analyses of APOE4 mice showed increased GLS activity in hippocampal CD11b+ enriched cells versus age-matched controls, which was completely normalized by JHU-083 treatment. JHU-083 was well-tolerated, showing no weight loss effect or overt behavioral changes. Peripheral nerve function, gastrointestinal histopathology, and CBC/clinical chemistry parameters were all unaffected by chronic JHU-083 treatment.

Conclusion: These results suggest that the attenuation of upregulated hippocampal glutaminase by JHU-083 represents a new therapeutic strategy for AD.
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http://dx.doi.org/10.3233/JAD-190588DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7678030PMC
January 2020

Dendrimer-conjugated glutaminase inhibitor selectively targets microglial glutaminase in a mouse model of Rett syndrome.

Theranostics 2020 27;10(13):5736-5748. Epub 2020 Apr 27.

Center for Nanomedicine, Department of Ophthalmology, Wilmer Eye Institute Johns Hopkins University School of Medicine, Baltimore MD, 21231.

: Elevated glutamate production and release from glial cells is a common feature of many CNS disorders. Inhibitors of glutaminase (GLS), the enzyme responsible for converting glutamine to glutamate have been developed to target glutamate overproduction. However, many GLS inhibitors have poor aqueous solubility, are unable to cross the blood brain barrier, or demonstrate significant toxicity when given systemically, precluding translation. Enhanced aqueous solubility and systemic therapy targeted to activated glia may address this challenge. Here we examine the impact of microglial-targeted GLS inhibition in a mouse model of Rett syndrome (RTT), a developmental disorder with no viable therapies, manifesting profound central nervous system effects, in which elevated glutamatergic tone, upregulation of microglial GLS, oxidative stress and neuroimmune dysregulation are key features. : To enable this, we conjugated a potent glutaminase inhibitor, -(5-{2-[2-(5-amino-[1,3,4]thiadiazol-2-yl)-ethylsulfanyl]-ethyl}-[1,3,4]thiadiazol-2-yl)-2-phenyl-acetamide (JHU29) to a generation 4 hydroxyl PAMAM dendrimer (D-JHU29). We then examined the effect of D-JHU29 in organotypic slice culture on glutamate release. We also examined GLS activity in microglial and non-microglial cells, and neurobehavioral phenotype after systemic administration of D-JHU29 in a mouse model of RTT. : We report successful conjugation of JHU29 to dendrimer resulting in enhanced water solubility compared to free JHU29. D-JHU29 reduced the excessive glutamate release observed in tissue culture slices in a clinically relevant -knockout (KO) RTT mouse. Microglia isolated from -KO mice demonstrated upregulation of GLS activity that normalized to wild-type levels following systemic treatment with D-JHU29. Neurobehavioral assessments in D-JHU29 treated -KO mice revealed selective improvements in mobility. : These findings demonstrate that glutaminase inhibitors conjugated to dendrimers are a viable mechanism to selectively inhibit microglial GLS to reduce glutamate production and improve mobility in a mouse model of RTT, with broader implications for selectively targeting this pathway in other neurodegenerative disorders.
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http://dx.doi.org/10.7150/thno.41714DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7254984PMC
April 2020

Dendrimer size effects on the selective brain tumor targeting in orthotopic tumor models upon systemic administration.

Bioeng Transl Med 2020 May 14;5(2):e10160. Epub 2020 Apr 14.

Center for Nanomedicine Wilmer Eye Institute, Johns Hopkins School of Medicine Baltimore Maryland USA.

Malignant gliomas are the most common and aggressive form of primary brain tumors, with a median survival of 15-20 months for patients receiving maximal interventions. Advances in nanomedicine have provided tumor-specific delivery of chemotherapeutics to potentially overcome their off-target toxicities. Recent advances in dendrimer-based nanomedicines have established that hydroxyl-terminated poly(amidoamine) dendrimers can intrinsically target neuroinflammation and brain tumors from systemic administration without the need for targeting moieties. The size of nanocarriers is a critical parameter that determines their tumor-targeting efficiency, intratumor distribution, and clearance mechanism. In this study, we explore the dendrimer size effects on brain tumor targeting capability in two clinically relevant orthotopic brain tumor models, the 9L rat and GL261 mouse models, which capture differing aspects of gliomas. We show that increasing dendrimers from Generation 4 to Generation 6 significantly enhances their tumor accumulation (~10-fold greater at 24 hr), tumor specificity (~2-3 fold higher), and tumor retention. The superior tumor targeting effect of G6 dendrimers is associated with its reduced renal clearance rate, resulting in longer circulation time compared to G4 dendrimers. Additionally, the increase in dendrimer generation does not compromise its homogeneous tumor distribution and intrinsic targeting of tumor-associated macrophages. These results validate the potential for these dendrimers as an effective, clinically translatable platform for effectively targeting tumor-associated macrophages in malignant gliomas.
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http://dx.doi.org/10.1002/btm2.10160DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7237147PMC
May 2020

Dendrimer mediated targeted delivery of sinomenine for the treatment of acute neuroinflammation in traumatic brain injury.

J Control Release 2020 07 24;323:361-375. Epub 2020 Apr 24.

Center for Nanomedicine, Department of Ophthalmology, Wilmer Eye Institute Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA; Department of Anesthesiology and Critical Care Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA; Hugo W. Moser Research Institute at Kennedy Krieger, Inc., Baltimore, MD 21205, USA. Electronic address:

Traumatic brain injury (TBI) is a significant medical problem with limited treatment options and is one of the main causes of life-long disability. Neuroinflammation orchestrated by activated microglia/macrophages at the site of injury plays a critical role in the onset of many pathological events following TBI, leading to blood brain barrier (BBB) dysfunction, neuronal damage and long term neuronal and behavioral deficits. Current treatment involves intravenous administration of anti-inflammatory drugs which have limited clinical outcomes only when dosed within the early time window after injury. Hence there is an urgent need to develop improved drug delivery systems which have potential to cross impaired BBB, target and deliver drugs selectively to activated microglia/macrophages at the sites of injury, and suppress the detrimental effects of acute inflammation. In this study, we have used Sinomenine (Sino), a potent anti-inflammatory and antioxidant drug conjugated to hydroxyl terminated generation-4 PAMAM dendrimer (D-Sino) as a potential therapy for attenuating early inflammation in TBI. D-Sino conjugates were synthesized using highly robust copper-catalyzed click reaction with high purity. D-Sino conjugates enhanced the intracellular availability of Sino due to their rapid cellular uptake, significantly attenuated early/acute inflammation by suppressing pro-inflammatory cytokines (TNF-α, IL-1β, CCL-3 and IL-6), and reduced oxidative stress (iNOS and NO) in LPS activated murine macrophages (RAW 264.7) by inhibiting NF-κB activation and its nuclear translocation (the root cause for inflammation inception) significantly more as compared to the free drug. Upon systemic administration in a rabbit model of pediatric TBI, D-Sino conjugates specifically targeted activated microglia/macrophages at the site of injury in the brain. Single dose of D-Sino attenuated inflammation in the injured brain areas by suppressing inflammatory cytokines expression whereas free Sino treatment did not demonstrate a significant effect. Together, these results suggest that D-Sino conjugate may open up new avenues for increasing the therapeutic window in the treatment of early inflammation and for improving the efficacy of the drug in TBI. Moreover, this treatment can work in conjunction with current clinical practices such as therapeutic hypothermia and pharmacologically induced coma for many indications associated with TBI, where acute inflammation plays a critical role in disease progression.
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http://dx.doi.org/10.1016/j.jconrel.2020.04.036DOI Listing
July 2020

Pediatric Traumatic Brain Injury Causes Long-Term Deficits in Adult Hippocampal Neurogenesis and Cognition.

J Neurotrauma 2020 Jul 17;37(14):1656-1667. Epub 2020 Apr 17.

Department of Anesthesiology and Critical Care Medicine, Johns Hopkins School of Medicine, Baltimore, Maryland, USA.

Young children who have sustained severe traumatic brain injury (TBI) can suffer from debilitating neurocognitive deficits. Impairment of adult hippocampal neurogenesis is associated with cognitive deficits and depression. Very few studies have investigated the adult hippocampal neurogenesis after pediatric TBI. Here, we evaluated long-term cognition, adult hippocampal neurogenesis, and microglial activation in a rabbit pediatric TBI model. On Post-natal Day 5-7 (P5-7), New Zealand white rabbits from the same litter were randomized into naïve, sham (craniotomy alone), and TBI (controlled cortical impact). Bromodeoxyuridine (BrdU, 50 mg/kg, intraperitoneally) was administered at 1-month post-injury, once/daily for 5 consecutive days. Novel object recognition and spontaneous alternation in T-maze tests were performed at 2 months post-injury to measure the cognitive functions. The animals were euthanized after behavioral tests at 3 months of age to evaluate adult hippocampal neurogenesis and microglial activation. We found that: 1) pediatric TBI caused significant deficits in hippocampal dependent cognitive functions; 2) the survival rates of adult-born neurons at both ipsilateral and contralateral hippocampus significantly decreased in the TBI group; 3) TBI induced ectopic migration of adult-born neurons at the dorsal dentate gyrus in both ipsilateral and contralateral hippocampus; 4) TBI increased astrogenesis in the hilus of the dentate gyrus; and 5) TBI results in abnormal microglial activation. In conclusion, pediatric TBI causes prolonged neuroinflammation and dysregulation of the adult hippocampal neurogenesis through young adulthood, which might be responsible for the cognitive deficits. Protection of adult hippocampal neurogenesis may potentially improve outcomes.
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http://dx.doi.org/10.1089/neu.2019.6894DOI Listing
July 2020

Dense hydroxyl polyethylene glycol dendrimer targets activated glia in multiple CNS disorders.

Sci Adv 2020 01 22;6(4):eaay8514. Epub 2020 Jan 22.

Center for Nanomedicine, Department of Ophthalmology, Wilmer Eye Institute Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA.

Poor transport of neuropharmaceutics through central nervous system (CNS) barriers limits the development of effective treatments for CNS disorders. We present the facile synthesis of a novel neuroinflammation-targeting polyethylene glycol-based dendrimer (PEGOL-60) using an efficient click chemistry approach. PEGOL-60 reduces synthetic burden by achieving high hydroxyl surface density at low generation, which plays a key role in brain penetration and glia targeting of dendrimers in CNS disorders. Systemically administered PEGOL-60 crosses impaired CNS barriers and specifically targets activated microglia/macrophages at the injured site in diverse animal models for cerebral palsy, glioblastoma, and age-related macular degeneration, demonstrating its potential to overcome impaired blood-brain, blood-tumor-brain, and blood-retinal barriers and target key cells in the CNS. PEGOL-60 also exhibits powerful intrinsic anti-oxidant and anti-inflammatory effects in inflamed microglia in vitro. Therefore, PEGOL-60 is an effective vehicle to specifically deliver therapies to sites of CNS injury for enhanced therapeutic outcomes in a range of neuroinflammatory diseases.
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http://dx.doi.org/10.1126/sciadv.aay8514DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6976300PMC
January 2020

Transient neonatal sleep fragmentation results in long-term neuroinflammation and cognitive impairment in a rabbit model.

Exp Neurol 2020 05 24;327:113212. Epub 2020 Jan 24.

Department of Anesthesiology and Critical Care Medicine, Johns Hopkins University School of Medicine, United States of America. Electronic address:

Sleep fragmentation is an increase in sleep-wake transitions without an overall decrease in total sleep time. Sleep fragmentation is well documented during acute and chronic hospitalization and can result in delirium and memory problems in children. Sleep fragmentation is also often noted in neurodevelopmental disorders. However, it is unclear how sleep fragmentation independent of disease affects brain development and function. We hypothesized that acute sleep fragmentation during the neonatal period in otherwise healthy animals would result in neuroinflammation and would be associated with abnormalities in cognitive development. The orbital shaker method was used to fragment sleep for 72 h in postnatal day 3 New Zealand white rabbit kits (fragmentation group). To control for maternal separation, the sham group was separated from the dam and maintained in the same conditions without undergoing sleep fragmentation. A naïve control group remained with the dam. Kits underwent behavioral testing with novel object recognition and spontaneous alternation T-maze tests at 2-3 weeks post-fragmentation and were sacrificed 3-50 days after fragmentation. Sleep fragmentation resulted in acute and chronic changes in microglial morphology in the hippocampus and cortex, and regional differences in mRNA expression of pro- and anti-inflammatory cytokines at 3, 7 and 50 days post-fragmentation. Impaired novel object recognition and a longer latency in T-maze task completion were noted in the fragmented kits. This was in spite of normalization of sleep architecture noted at 2 months of age in these kits. The results indicate that transient neonatal sleep fragmentation results in short-term and long-term immune alterations in the brain, along with diminished performance in cognitive tasks long-term.
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http://dx.doi.org/10.1016/j.expneurol.2020.113212DOI Listing
May 2020

Nanotechnology enabled regenerative medicine for neurological disorders.

Adv Drug Deliv Rev 2019 08;148:1-2

Department of Anesthesiology and Critical Care Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA.

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http://dx.doi.org/10.1016/j.addr.2019.11.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7474548PMC
August 2019

Advanced nanotherapies to promote neuroregeneration in the injured newborn brain.

Adv Drug Deliv Rev 2019 08 31;148:19-37. Epub 2019 Oct 31.

Department of Pediatrics, University of California San Francisco School of Medicine, San Francisco, CA, United States; Department of Neurology, University of California San Francisco School of Medicine, San Francisco, CA, United States. Electronic address:

Neonatal brain injury affects thousands of babies each year and may lead to long-term and permanent physical and neurological problems. Currently, therapeutic hypothermia is standard clinical care for term newborns with moderate to severe neonatal encephalopathy. Nevertheless, it is not completely protective, and additional strategies to restore and promote regeneration are urgently needed. One way to ensure recovery following injury to the immature brain is to augment endogenous regenerative pathways. However, novel strategies such as stem cell therapy, gene therapies and nanotechnology have not been adequately explored in this unique age group. In this perspective review, we describe current efforts that promote neuroprotection and potential targets that are unique to the developing brain, which can be leveraged to facilitate neuroregeneration.
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http://dx.doi.org/10.1016/j.addr.2019.10.005DOI Listing
August 2019

Neuronanotechnology for brain regeneration.

Adv Drug Deliv Rev 2019 08 17;148:3-18. Epub 2019 Apr 17.

Department of Anesthesiology and Critical Care Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21287, United States of America. Electronic address:

Identifying and harnessing regenerative pathways while suppressing the growth-inhibiting processes of the biological response to injury is the central goal of stimulating neurogenesis after central nervous system (CNS) injury. However, due to the complexity of the mature CNS involving a plethora of cellular pathways and extracellular cues, as well as difficulties in accessibility without highly invasive procedures, clinical successes of regenerative medicine for CNS injuries have been extremely limited. Current interventions primarily focus on stabilization and mitigation of further neuronal death rather than direct stimulation of neurogenesis. In the past few decades, nanotechnology has offered substantial innovations to the field of regenerative medicine. Their nanoscale features allow for the fine tuning of biological interactions for enhancing drug delivery and stimulating cellular processes. This review gives an overview of nanotechnology applications in CNS regeneration organized according to cellular and extracellular targets and discuss future directions for the field.
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http://dx.doi.org/10.1016/j.addr.2019.04.004DOI Listing
August 2019

Administration of a 20-Hydroxyeicosatetraenoic Acid Synthesis Inhibitor Improves Outcome in a Rat Model of Pediatric Traumatic Brain Injury.

Dev Neurosci 2019 25;41(3-4):166-176. Epub 2019 Sep 25.

Department of Anesthesiology and Critical Care Medicine, Johns Hopkins University, School of Medicine, Baltimore, Maryland, USA,

The arachidonic acid pathway metabolite 20-hydroxyeicosatetraenoic acid (20-HETE) contributes to ischemia/reperfusion brain injury. Inhibition of 20-HETE formation can protect the developing brain from global ischemia. Here, we examined whether treatment with the 20-HETE synthesis inhibitor N-hydroxy-N-4-butyl-2-methylphenylformamidine (HET0016) can protect the immature brain from traumatic brain injury (TBI). Male rats at postnatal day 9-10 underwent controlled cortical impact followed by intraperitoneal injection with vehicle or HET0016 (1 mg/kg, 5 min and 3 h post-injury). HET0016 decreased the lesion volume by over 50% at 3 days of recovery, and this effect persisted at 30 days as the brain matured. HET0016 decreased peri-lesion gene expression of proinflammatory cytokines (tumor necrosis factor-α [TNF-α], interleukin-1β [IL-1β]) at 1 day and increased reparative cytokine (IL-4, IL-10) expression at 3 days. It also partially preserved microglial ramified processes, consistent with less activation. HET0016 decreased contralateral hindlimb foot faults and improved outcome on the novel object recognition memory task 30 days after TBI. In cultured BV2 microglia, HET0016 attenuated the lipopolysaccharide-evoked increase in release of TNF-α. Our data show that HET0016 improves acute and long-term histologic and functional outcomes, in association with an attenuated neuroinflammatory response after contusion of an immature rat brain.
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http://dx.doi.org/10.1159/000500895DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7044071PMC
June 2020

Neuroimmune responses in the developing brain following traumatic brain injury.

Exp Neurol 2019 10 17;320:112957. Epub 2019 May 17.

Department of Anesthesiology and Critical Care Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21287, United States of America. Electronic address:

Traumatic brain injury (TBI) is one of the leading causes of both acute and long-term morbidity in the pediatric population, leading to a substantial, long-term socioeconomic burden. Despite the increase in the amount of pre-clinical and clinical research, treatment options for TBI rely heavily on supportive care with very limited targeted interventions that improve the acute and chronic sequelae of TBI. Other than injury prevention, not much can be done to limit the primary injury, which consists of tissue damage and cellular destruction. Secondary injury is the result of the ongoing complex inflammatory pathways that further exacerbate tissue damage, resulting in the devastating chronic outcomes of TBI. On the other hand, some level of inflammation is essential for neuronal regeneration and tissue repair. In this review article we discuss the various stages of the neuroimmune response in the immature, pediatric brain in the context of normal maturation and development of the immune system. The developing brain has unique features that distinguish it from the adult brain, and the immune system plays an integral role in CNS development. Those features could potentially make the developing brain more susceptible to worse outcomes, both acutely and in the long-term. The neuroinflammatory reaction which is triggered by TBI can be described as a highly intricate interaction between the cells of the innate and the adaptive immune systems. The innate immune system is triggered by non-specific danger signals that are released from damaged cells and tissues, which in turn leads to neutrophil infiltration, activation of microglia and astrocytes, complement release, as well as histamine release by mast cells. The adaptive immune response is subsequently activated leading to the more chronic effects of neuroinflammation. We will also discuss current attempts at modulating the TBI-induced neuroinflammatory response. A better understanding of the role of the immune system in normal brain development and how immune function changes with age is crucial for designing therapies to appropriately target the immune responses following TBI in order to enhance repair and plasticity.
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http://dx.doi.org/10.1016/j.expneurol.2019.112957DOI Listing
October 2019

Targeting Mitochondrial Dysfunction and Oxidative Stress in Activated Microglia using Dendrimer-Based Therapeutics.

Theranostics 2018 5;8(20):5529-5547. Epub 2018 Nov 5.

Center for Nanomedicine, Department of Ophthalmology, Wilmer Eye Institute Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA.

Mitochondrial oxidative stress is associated with many neurodegenerative diseases, such as traumatic brain injury (TBI). Targeted delivery of antioxidants to mitochondria has failed to translate into clinical success due to their nonspecific cellular localization, poor transport properties across multiple biological barriers, and associated side effects. These challenges, coupled with the complex function of the mitochondria, create the need for innovative delivery strategies. Neutral hydroxyl-terminated polyamidoamine (PAMAM) dendrimers have shown significant potential as nanocarriers in multiple brain injury models. -acetyl cysteine (NAC) is a clinically used antioxidant and anti-inflammatory agent which has shown significant potency when delivered in a targeted manner. Here we present a mitochondrial targeting hydroxyl PAMAM dendrimer-drug construct (TPP-D-NAC) with triphenyl-phosphonium (TPP) for mitochondrial targeting and NAC for targeted delivery to mitochondria in injured glia. Co-localization and mitochondrial content of mitochondria-targeted and unmodified dendrimer were assessed in microglia and macrophages via immunohistochemistry and fluorescence quantification. Therapeutic improvements of TPP-D-NAC over dendrimer-NAC conjugate (D-NAC) and free NAC were evaluated in microglia under oxidative stress challenge. neuroinflammation targeting was confirmed in a rabbit model of TBI. TPP-conjugated dendrimer co-localized significantly more with mitochondria than unmodified dendrimer without altering overall levels of cellular internalization. This targeting capability translated to significant improvements in the attenuation of oxidative stress by TPP-D-NAC compared to D-NAC and free NAC. Upon systemic administration in a rabbit TBI model, TPP-conjugated dendrimer co-localized specifically with mitochondria in activated microglia and macrophages in the white matter of the ipsilateral/injured hemisphere, confirming its BBB penetration and glial targeting capabilities. D-NAC has shown promising efficacy in many animal models of neurodegeneration, and this work provides evidence that modification for mitochondrial targeting can further enhance its therapeutic efficacy, particularly in diseases where oxidative stress-induced glial cell death plays a significant role in disease progression.
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http://dx.doi.org/10.7150/thno.29039DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6276292PMC
September 2019

Cognitive impairments induced by necrotizing enterocolitis can be prevented by inhibiting microglial activation in mouse brain.

Sci Transl Med 2018 12;10(471)

Division of General Pediatric Surgery, Johns Hopkins University and Bloomberg Children's Center, Johns Hopkins Hospital, Baltimore, MD 21287, USA.

Necrotizing enterocolitis (NEC) is a severe gastrointestinal disease of the premature infant. One of the most important long-term complications observed in children who survive NEC early in life is the development of profound neurological impairments. However, the pathways leading to NEC-associated neurological impairments remain unknown, thus limiting the development of prevention strategies. We have recently shown that NEC development is dependent on the expression of the lipopolysaccharide receptor Toll-like receptor 4 (TLR4) on the intestinal epithelium, whose activation by bacteria in the newborn gut leads to mucosal inflammation. Here, we hypothesized that damage-induced production of TLR4 endogenous ligands in the intestine might lead to activation of microglial cells in the brain and promote cognitive impairments. We identified a gut-brain signaling axis in an NEC mouse model in which activation of intestinal TLR4 signaling led to release of high-mobility group box 1 in the intestine that, in turn, promoted microglial activation in the brain and neurological dysfunction. We further demonstrated that an orally administered dendrimer-based nanotherapeutic approach to targeting activated microglia could prevent NEC-associated neurological dysfunction in neonatal mice. These findings shed light on the molecular pathways leading to the development of NEC-associated brain injury, provide a rationale for early removal of diseased intestine in NEC, and indicate the potential of targeted therapies that protect the developing brain in the treatment of NEC in early childhood.
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http://dx.doi.org/10.1126/scitranslmed.aan0237DOI Listing
December 2018

Noninvasive C-rifampin positron emission tomography reveals drug biodistribution in tuberculous meningitis.

Sci Transl Med 2018 12;10(470)

Center for Infection and Inflammation Imaging Research, Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA.

Tuberculous meningitis (TBM) is a devastating form of tuberculosis (TB), and key TB antimicrobials, including rifampin, have restricted brain penetration. A lack of reliable data on intralesional drug biodistribution in infected tissues has limited pharmacokinetic (PK) modeling efforts to optimize TBM treatments. Current methods to measure intralesional drug distribution rely on tissue resection, which is difficult in humans and generally limited to a single time point even in animals. In this study, we developed a multidrug treatment model in rabbits with experimentally induced TBM and performed serial noninvasive dynamic C-rifampin positron emission tomography (PET) over 6 weeks. Area under the curve brain/plasma ratios were calculated using PET and correlated with postmortem mass spectrometry. We demonstrate that rifampin penetration into infected brain lesions is limited, spatially heterogeneous, and decreases rapidly as early as 2 weeks into treatment. Moreover, rifampin concentrations in the cerebrospinal fluid did not correlate well with those in the brain lesions. First-in-human C-rifampin PET performed in a patient with TBM confirmed these findings. PK modeling predicted that rifampin doses (≥30 mg/kg) were required to achieve adequate intralesional concentrations in young children with TBM. These data demonstrate the proof of concept of PET as a clinically translatable tool to noninvasively measure intralesional antimicrobial distribution in infected tissues.
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http://dx.doi.org/10.1126/scitranslmed.aau0965DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6360528PMC
December 2018

Altered trajectories of neurodevelopment and behavior in mouse models of Rett syndrome.

Neurobiol Learn Mem 2019 11 29;165:106962. Epub 2018 Nov 29.

Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA; The Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA; The Hugo W. Moser Research Institute at Kennedy Krieger, Inc., Baltimore, MD 21205, USA. Electronic address:

Rett Syndrome (RTT) is a genetic disorder that is caused by mutations in the x-linked gene coding for methyl-CpG-biding-protein 2 (MECP2) and that mainly affects females. Male and female transgenic mouse models of RTT have been studied extensively, and we have learned a great deal regarding RTT neuropathology and how MeCP2 deficiency may be influencing brain function and maturation. In this manuscript we review what is known concerning structural and coinciding functional and behavioral deficits in RTT and in mouse models of MeCP2 deficiency. We also introduce our own corroborating data regarding behavioral phenotype and morphological alterations in volume of the cortex and striatum and the density of neurons, aberrations in experience-dependent plasticity within the barrel cortex and the impact of MeCP2 loss on glial structure. We conclude that regional structural changes in genetic models of RTT show great similarity to the alterations in brain structure of patients with RTT. These region-specific modifications often coincide with phenotype onset and contribute to larger issues of circuit connectivity, progression, and severity. Although the alterations seen in mouse models of RTT appear to be primarily due to cell-autonomous effects, there are also non-cell autonomous mechanisms including those caused by MeCP2-deficient glia that negatively impact healthy neuronal function. Collectively, this body of work has provided a solid foundation on which to continue to build our understanding of the role of MeCP2 on neuronal and glial structure and function, its greater impact on neural development, and potential new therapeutic avenues.
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http://dx.doi.org/10.1016/j.nlm.2018.11.007DOI Listing
November 2019

Dendrimer-N-acetyl-L-cysteine modulates monophagocytic response in adrenoleukodystrophy.

Ann Neurol 2018 09;84(3):452-462

Moser Center for Leukodystrophies, Kennedy Krieger Institute.

Objective: X-linked adrenoleukodystrophy (ALD) is a neurodegenerative disorder due to mutations in the peroxisomal very long-chain fatty acyl-CoA transporter, ABCD1, with limited therapeutic options. ALD may manifest in a slowly progressive adrenomyeloneuropathy (AMN) phenotype, or switch to rapid inflammatory demyelinating cerebral disease (cALD), in which microglia have been shown to play a pathophysiological role. The aim of this study was to determine the role of patient phenotype in the immune response of ex vivo monophagocytic cells to stimulation, and to evaluate the efficacy of polyamidoamine dendrimer conjugated to the antioxidant precursor N-acetyl-cysteine (NAC) in modulating this immune response.

Methods: Human monophagocytic cells were derived from fresh whole blood, from healthy (n = 4), heterozygote carrier (n = 4), AMN (n = 7), and cALD (n = 4) patients. Cells were exposed to very long-chain fatty acids (VLCFAs; C24:0 and C26:0) and treated with dendrimer-NAC (D-NAC).

Results: Ex vivo exposure to VLCFAs significantly increased tumor necrosis factor α (TNFα) and glutamate secretion from cALD patient macrophages. Additionally, a significant reduction in total intracellular glutathione was observed in cALD patient cells. D-NAC treatment dose-dependently reduced TNFα and glutamate secretion and replenished total intracellular glutathione levels in cALD patient macrophages, more efficiently than NAC. Similarly, D-NAC treatment decreased glutamate secretion in AMN patient cells.

Interpretation: ALD phenotypes display unique inflammatory profiles in response to VLCFA stimulation, and therefore ex vivo monophagocytic cells may provide a novel test bed for therapeutic agents. Based on our findings, D-NAC may be a viable therapeutic strategy for the treatment of cALD. Ann Neurol 2018;84:452-462.
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http://dx.doi.org/10.1002/ana.25303DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6454885PMC
September 2018

Scalable synthesis and validation of PAMAM dendrimer--acetyl cysteine conjugate for potential translation.

Bioeng Transl Med 2018 May 25;3(2):87-101. Epub 2018 May 25.

Center for Nanomedicine, Department of Ophthalmology Wilmer Eye Institute Johns Hopkins University School of Medicine Baltimore MD 21287.

Dendrimer--acetyl cysteine (D-NAC) conjugate has shown significant promise in multiple preclinical models of brain injury and is undergoing clinical translation. D-NAC is a generation-4 hydroxyl-polyamidoamine dendrimer conjugate where -acetyl cysteine (NAC) is covalently bound through disulfide linkages on the surface of the dendrimer. It has shown remarkable potential to selectively target and deliver NAC to activated microglia and astrocytes at the site of brain injury in several animal models, producing remarkable improvements in neurological outcomes at a fraction of the free drug dose. Here we present a highly efficient, scalable, greener, well-defined route to the synthesis of D-NAC, and validate the structure, stability and activity to define the benchmarks for this compound. This newly developed synthetic route has significantly reduced the synthesis time from three weeks to one week, uses industry-friendly solvents/reagents, and involves simple purification procedures, potentially enabling efficient scale up.
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http://dx.doi.org/10.1002/btm2.10094DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6063872PMC
May 2018

Traumatic Injury Leads to Inflammation and Altered Tryptophan Metabolism in the Juvenile Rabbit Brain.

J Neurotrauma 2018 Sep 11. Epub 2018 Sep 11.

Department of Anesthesiology and Critical Care Medicine, Johns Hopkins University School f Medicine , Baltimore, Maryland.

Neuroinflammation after traumatic brain injury (TBI) contributes to widespread cell death and tissue loss. Here, we evaluated sequential inflammatory response in the brain, as well as inflammation-induced changes in brain tryptophan metabolism over time, in a rabbit pediatric TBI model. On post-natal days 5-7 (P5-P7), New Zealand white rabbit littermates were randomized into three groups: naïve (no injury), sham (craniotomy alone), and TBI (controlled cortical impact). Animals were sacrificed at 6 h and 1, 3, 7, and 21 days post-injury for evaluating levels of pro- and anti-inflammatory cytokines, as well as the major components in the tryptophan-kynurenine pathway. We found that 1) pro- and anti-inflammatory cytokine levels in the brain injury area were differentially regulated in a time-dependent manner post-injury; 2) indoleamine 2,3 dioxygeenase 1 (IDO1) was upregulated around the injury area in TBI kits that persisted at 21 days post-injury; 3) mean length of serotonin-staining fibers was significantly reduced in the injured brain region in TBI kits for at least 21 days post-injury; and 4) kynurenine level significantly increased at 7 days post-injury. A significant decrease in serotonin/tryptophan ratio and melatonin/tryptophan ratio at 21 days post-injury was noted, suggesting that tryptophan metabolism is altered after TBI. A better understanding of the temporal evolution of immune responses and tryptophan metabolism during injury and repair after TBI is crucial for the development of novel therapeutic strategies targeting these pathways.
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http://dx.doi.org/10.1089/neu.2017.5450DOI Listing
September 2018

Cerebellar injury and impaired function in a rabbit model of maternal inflammation induced neonatal brain injury.

Neurobiol Learn Mem 2019 11 24;165:106901. Epub 2018 Jul 24.

Department of Anesthesiology and Critical Care Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, USA. Electronic address:

Cerebellum is involved in higher cognitive functions and plays important roles in neurological disorders. Cerebellar injury has been detected frequently in patients with preterm birth resulting in cognitive dysfunction later in life. Maternal infection and inflammation is associated with preterm birth and in neonatal brain injury. We have previously shown that intrauterine lipopolysaccharide (LPS) exposure induces white matter injury and microglial activation in the cerebral white matter tracts of neonatal rabbits, resulting in motor deficits consistent with the clinical findings of cerebral palsy (CP). Here we investigated whether intrauterine LPS exposure induced cerebellar inflammation and functional impairment. Timed-pregnant New Zealand white rabbits underwent a laparotomy on gestational day 28 (G28) and LPS (3200 EU, endotoxin group) was injected along the wall of the uterus as previously described. Controls did not receive surgical intervention. Kits born to control and endotoxin treated dams were euthanized on postnatal day (PND)1 (3 days post-injury) or PND5 (7 days post-injury) and cerebellum evaluated for presence of inflammation. The microglial morphology in cerebellar white matter areas was analyzed using Neurolucida and Neurolucida Explorer. mRNA expression of inflammatory cytokines was quantified by real-time-PCR. We found that intrauterine exposure to LPS induced intensive microglial activation in cerebellar white matter areas, as evidenced by increased numbers of activated microglia and morphological changes (amoeboid soma and retracted processes) that was accompanied by significant increases in pro-inflammatory cytokines. The Purkinje cell layer was less developed in endotoxin exposed kits than healthy controls. In kits that survived to PND 60, soma size and cell density of Purkinje cells were significantly decreased in endotoxin exposed kits compared to controls. The findings of altered Purkinje cell morphology were consistent with impaired cerebellar function as tested by eye-blink conditioning at 1 month of age. The results indicate that the cerebellum is vulnerable to perinatal insults and that therapies targeting cerebellar inflammation and injury may help in improving outcomes and function.
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http://dx.doi.org/10.1016/j.nlm.2018.07.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6715541PMC
November 2019

Effect of mannose targeting of hydroxyl PAMAM dendrimers on cellular and organ biodistribution in a neonatal brain injury model.

J Control Release 2018 08 5;283:175-189. Epub 2018 Jun 5.

Center for Nanomedicine, Department of Ophthalmology, Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA; Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD 21218, USA; Hugo W. Moser Research Institute at Kennedy Krieger, Inc., Baltimore, MD 21205, USA; Kennedy Krieger Institute - Johns Hopkins University for Cerebral Palsy Research Excellence, Baltimore, MD 21218, USA. Electronic address:

Neurotherapeutics for the treatment of central nervous system (CNS) disorders must overcome challenges relating to the blood-brain barrier (BBB), brain tissue penetration, and the targeting of specific cells. Neuroinflammation mediated by activated microglia is a major hallmark of several neurological disorders, making these cells a desirable therapeutic target. Building on the promise of hydroxyl-terminated generation four polyamidoamine (PAMAM) dendrimers (D4-OH) for penetrating the injured BBB and targeting activated glia, we explored if conjugation of targeting ligands would enhance and modify brain and organ uptake. Since mannose receptors [cluster of differentiation (CD) 206] are typically over-expressed on injured microglia, we conjugated mannose to the surface of multifunctional D4-OH using highly efficient, atom-economical, and orthogonal Cu(I)-catalyzed alkyne-azide cycloaddition (CuAAC) click chemistry and evaluated the effect of mannose conjugation on the specific cell uptake of targeted and non-targeted dendrimers both in vitro and in vivo. In vitro results indicate that the conjugation of mannose as a targeting ligand significantly changes the mechanism of dendrimer internalization, giving mannosylated dendrimer a preference for mannose receptor-mediated endocytosis as opposed to non-specific fluid phase endocytosis. We further investigated the brain uptake and biodistribution of targeted and non-targeted fluorescently labeled dendrimers in a maternal intrauterine inflammation-induced cerebral palsy (CP) rabbit model using quantification methods based on fluorescence spectroscopy and confocal microscopy. We found that the conjugation of mannose modified the distribution of D4-OH throughout the body in this neonatal rabbit CP model without lowering the amount of dendrimer delivered to injured glia in the brain, even though significantly higher glial uptake was not observed in this model. Mannose conjugation to the dendrimer modifies the dendrimer's interaction with cells, but does not minimize its inherent inflammation-targeting abilities.
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http://dx.doi.org/10.1016/j.jconrel.2018.06.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6091673PMC
August 2018

Early Detection of Hypothermic Neuroprotection Using T2-Weighted Magnetic Resonance Imaging in a Mouse Model of Hypoxic Ischemic Encephalopathy.

Front Neurol 2018 8;9:304. Epub 2018 May 8.

Hugo W. Moser Research Institute at Kennedy Krieger, Baltimore, MD, United States.

Perinatal hypoxic-ischemic encephalopathy (HIE) can lead to neurodevelopmental disorders, including cerebral palsy. Standard care for neonatal HIE includes therapeutic hypothermia, which provides partial neuroprotection; magnetic resonance imaging (MRI) is often used to assess injury and predict outcome after HIE. Immature rodent models of HIE are used to evaluate mechanisms of injury and to examine the efficacy and mechanisms of neuroprotective interventions such as hypothermia. In this study, we first confirmed that, in the CD1 mouse model of perinatal HIE used for our research, MRI obtained 3 h after hypoxic ischemia (HI) could reliably assess initial brain injury and predict histopathological outcome. Mice were subjected to HI (unilateral carotid ligation followed by exposure to hypoxia) on postnatal day 7 and were imaged with T2-weighted MRI and diffusion-weighted MRI (DWI), 3 h after HI. Clearly defined regions of increased signal were comparable in T2 MRI and DWI, and we found a strong correlation between T2 MRI injury scores 3 h after HI and histopathological brain injury 7 days after HI, validating this method for evaluating initial injury in this model of HIE. The more efficient, higher resolution T2 MRI was used to score initial brain injury in subsequent studies. In mice treated with hypothermia, we found a significant reduction in T2 MRI injury scores 3 h after HI, compared to normothermic littermates. Early hypothermic neuroprotection was maintained 7 days after HI, in both T2 MRI injury scores and histopathology. In the normothermic group, T2 MRI injury scores 3 h after HI were comparable to those obtained 7 days after HI. However, in the hypothermic group, brain injury was significantly less 7 days after HI than at 3 h. Thus, early neuroprotective effects of hypothermia were enhanced by 7 days, which may reflect the additional 3 h of hypothermia after imaging or effects on later mechanisms of injury, such as delayed cell death and inflammation. Our results demonstrate that hypothermia has early neuroprotective effects in this model. These findings suggest that hypothermia has an impact on early mechanisms of excitotoxic injury and support initiation of hypothermic intervention as soon as possible after diagnosis of HIE.
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http://dx.doi.org/10.3389/fneur.2018.00304DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5951924PMC
May 2018

Preferential and Increased Uptake of Hydroxyl-Terminated PAMAM Dendrimers by Activated Microglia in Rabbit Brain Mixed Glial Culture.

Molecules 2018 Apr 27;23(5). Epub 2018 Apr 27.

Department of Anesthesiology and Critical Care Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.

Polyamidoamine (PAMAM) dendrimers are multifunctional nanoparticles with tunable physicochemical features, making them promising candidates for targeted drug delivery in the central nervous system (CNS). Systemically administered dendrimers have been shown to localize in activated glial cells, which mediate neuroinflammation in the CNS. These dendrimers delivered drugs specifically to activated microglia, producing significant neurological improvements in multiple brain injury models, including in a neonatal rabbit model of cerebral palsy. To gain further insight into the mechanism of dendrimer cell uptake, we utilized an in vitro model of primary glial cells isolated from newborn rabbits to assess the differences in hydroxyl-terminated generation 4 PAMAM dendrimer (D4-OH) uptake by activated and non-activated glial cells. We used fluorescently-labelled D4-OH (D-Cy5) as a tool for investigating the mechanism of dendrimer uptake. D4-OH PAMAM dendrimer uptake was determined by fluorescence quantification using confocal microscopy and flow cytometry. Our results indicate that although microglial cells in the mixed cell population demonstrate early uptake of dendrimers in this in vitro system, activated microglia take up more dendrimer compared to resting microglia. Astrocytes showed delayed and limited uptake. We also illustrated the differences in mechanism of uptake between resting and activated microglia using different pathway inhibitors. Both resting and activated microglia primarily employed endocytotic pathways, which are enhanced in activated microglial cells. Additionally, we demonstrated that hydroxyl terminated dendrimers are taken up by primary microglia using other mechanisms including pinocytosis, caveolae, and aquaporin channels for dendrimer uptake.
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http://dx.doi.org/10.3390/molecules23051025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6102539PMC
April 2018