Multimodal assessment of non-alcoholic fatty liver disease with transmission-reflection optoacoustic ultrasound.

Non-alcoholic fatty liver disease (NAFLD) is an umbrella term referring to a group of conditions associated to fat deposition and damage of liver tissue. Early detection of fat accumulation is essential to avoid progression of NAFLD to serious pathological stages such as liver cirrhosis and hepatocellular carcinoma. Methods: We exploited the unique capabilities of transmission-reflection optoacoustic ultrasound (TROPUS), which combines the advantages of optical and acoustic contrasts, for an early-stage multi-parametric assessment of NAFLD in mice. Results: The multispectral optoacoustic imaging allowed for spectroscopic differentiation of lipid content, as well as the bio-distributions of oxygenated and deoxygenated hemoglobin in liver tissues in vivo. The pulse-echo (reflection) ultrasound (US) imaging further provided a valuable anatomical reference whilst transmission US facilitated the mapping of speed of sound changes in lipid-rich regions, which was consistent with the presence of macrovesicular hepatic steatosis in the NAFLD livers examined with ex vivo histological staining. Conclusion: The proposed multimodal approach facilitates quantification of liver abnormalities at early stages using a variety of optical and acoustic contrasts, laying the ground for translating the TROPUS approach toward diagnosis and monitoring NAFLD in patients.

Optoacoustic characterization of breast conserving surgery specimens - A pilot study.

In this pilot study, we tested an ultrasound-guided optoacoustic tomography (US-OT) two-dimensional (2D) array scanner to understand the optoacoustic patterns of excised breastconserving surgery (BCS) specimens. We imaged 14 BCS specimens containing malignant tumors at eight wavelengths spanning 700-1100 nm. Spectral unmixing across multiple wavelengths allowed for visualizing major intrinsic chromophores in the breast tissue including hemoglobin and lipid up to a depth of 7 mm. We identified less/no lipid signals within the tumor and intense deoxy-hemoglobin (Hb) signals on the rim of the tumor as unique characteristics of malignant tumors in comparison to no tumor region. We also observed continuous broad lipid signals as features of negative margins and compromised lipid signals interrupted by vasculature as features of positive margins. These differentiating patterns can form the basis of US-OT to be explored as an alternate, fast and efficient intraoperative method for evaluation of tumor resection margins.

Ultrasound Guided Optoacoustic Tomography in Assessment of Tumor Margins for Lumpectomies.

PURPOSE: To determine the accuracy of a handheld ultrasound-guided optoacoustic tomography (US-OT) probe developed for human deep-tissue imaging in ex vivo assessment of tumor margins postlumpectomy. METHODS: A custom-built two-dimensional (2D) US-OT-handheld probe was used to scan 15 lumpectomy breast specimens. Optoacoustic signals acquired at multiple wavelengths between 700 and 1100 nm were reconstructed using model linear algorithm, followed by spectral unmixing for lipid and deoxyhemoglobin (Hb). Distribution maps of lipid and Hb on the anterior, posterior, superior, inferior, medial, and lateral margins of the specimens were inspected for margin involvement, and results were correlated with histopathologic findings. The agreement in tumor margin assessment between US-OT and histopathology was determined using the Bland-Altman plot. Accuracy, sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of margin assessment using US-OT were calculated. RESULTS: Ninety margins (6 × 15 specimens) were assessed. The US-OT probe resolved blood vessels and lipid up to a depth of 6 mm. Negative and positive margins were discriminated by marked differences in the distribution patterns of lipid and Hb. US-OT assessments were concordant with histopathologic findings in 87 of 89 margins assessed (one margin was uninterpretable and excluded), with diagnostic accuracy of 97.9% (kappa = 0.79). The sensitivity, specificity, PPV, and NPV were 100% (4/4), 97.6% (83/85), 66.7% (4/6), and 100% (83/83), respectively. CONCLUSION: US-OT was capable of providing distribution maps of lipid and Hb in lumpectomy specimens that predicted tumor margins with high sensitivity and specificity, making it a potential tool for intraoperative tumor margin assessment.

Integrin-targeted quantitative optoacoustic imaging with MRI correlation for monitoring a BRAF/MEK inhibitor combination therapy in a murine model of human melanoma.

PURPOSE: To investigate αvß3-integrin-targeted optoacoustic imaging and MRI for monitoring a BRAF/MEK inhibitor combination therapy in a murine model of human melanoma. MATERIALS AND METHODS: Human BRAF V600E-positive melanoma xenograft (A375)-bearing Balb/c nude mice (n = 10) were imaged before (day 0) and after (day 7) a BRAF/MEK inhibitor combination therapy (encorafenib, 1.3 mg/kg/d; binimetinib, 0.6 mg/kg/d, n = 5) or placebo (n = 5), respectively. Optoacoustic imaging was performed on a preclinical system unenhanced and 5 h after i. v. injection of an αvß3-integrin-targeted fluorescent probe. The αvß3-integrin-specific tumor signal was derived by spectral unmixing. For morphology-based tumor response assessments, T2w MRI data sets were acquired on a clinical 3 Tesla scanner. The imaging results were validated by multiparametric immunohistochemistry (ß3 -integrin expression, CD31 -microvascular density, Ki-67 -proliferation). RESULTS: The αvß3-integrin-specific tumor signal was significantly reduced under therapy, showing a unidirectional decline in all animals (from 7.98±2.22 to 1.67±1.30; p = 0.043). No significant signal change was observed in the control group (from 6.60±6.51 to 3.67±1.93; p = 0.500). Immunohistochemistry revealed a significantly lower integrin expression (ß3: 0.20±0.02 vs. 0.39±0.05; p = 0.008) and microvascular density (CD31: 119±15 vs. 292±49; p = 0.008) in the therapy group. Tumor volumes increased with no significant intergroup difference (therapy: +107±42 mm3; control +112±44mm3, p = 0.841). In vivo blocking studies with αvß3-integrin antagonist cilengitide confirmed the target specificity of the fluorescent probe. CONCLUSIONS: αvß3-integrin-targeted optoacoustic imaging allowed for the early non-invasive monitoring of a BRAF/MEK inhibitor combination therapy in a murine model of human melanoma, adding molecular information on tumor receptor status to morphology-based tumor response criteria.

Dynamic and accurate assessment of acetaminophen-induced hepatotoxicity by integrated photoacoustic imaging and mechanistic biomarkers in vivo.

The prediction and understanding of acetaminophen (APAP)-induced liver injury (APAP-ILI) and the response to therapeutic interventions is complex. This is due in part to sensitivity and specificity limitations of currently used assessment techniques. Here we sought to determine the utility of integrating translational non-invasive photoacoustic imaging of liver function with mechanistic circulating biomarkers of hepatotoxicity with histological assessment to facilitate the more accurate and precise characterization of APAP-ILI and the efficacy of therapeutic intervention. Perturbation of liver function and cellular viability was assessed in C57BL/6J male mice by Indocyanine green (ICG) clearance (Multispectral Optoacoustic Tomography (MSOT)) and by measurement of mechanistic (miR-122, HMGB1) and established (ALT, bilirubin) circulating biomarkers in response to the acetaminophen and its treatment with acetylcysteine (NAC) in vivo. We utilised a 60% partial hepatectomy model as a situation of defined hepatic functional mass loss to compared acetaminophen-induced changes to. Integration of these mechanistic markers correlated with histological features of APAP hepatotoxicity in a time-dependent manner. They accurately reflected the onset and recovery from hepatotoxicity compared to traditional biomarkers and also reported the efficacy of NAC with high sensitivity. ICG clearance kinetics correlated with histological scores for acute liver damage for APAP (i.e. 3h timepoint; r=0.90, P<0.0001) and elevations in both of the mechanistic biomarkers, miR-122 (e.g. 6h timepoint; r=0.70, P=0.005) and HMGB1 (e.g. 6h timepoint; r=0.56, P=0.04). For the first time we report the utility of this non-invasive longitudinal imaging approach to provide direct visualisation of the liver function coupled with mechanistic biomarkers, in the same animal, allowing the investigation of the toxicological and pharmacological aspects of APAP-ILI and hepatic regeneration.

Constrained Inversion and Spectral Unmixing in Multispectral Optoacoustic Tomography.

Accurate extraction of physical and biochemical parameters from optoacoustic images is often impeded due to the use of unrigorous inversion schemes, incomplete tomographic detection coverage, or other experimental factors that cannot be readily accounted for during the image acquisition and reconstruction process. For instance, inaccurate assumptions in the physical forward model may lead to negative optical absorption values in the reconstructed images. Any artifacts present in the single wavelength optoacoustic images can be significantly aggravated when performing a two-step reconstruction consisting in acoustic inversion and spectral unmixing aimed at rendering the distributions of spectrally distinct absorbers. We investigate a number of algorithmic strategies with non-negativity constraints imposed at the different phases of the reconstruction process. Performance is evaluated in cross-sectional multispectral optoacoustic tomography recordings from tissue-mimicking phantoms and in vivo mice embedded with varying concentrations of contrast agents. Additional in vivo validation is subsequently performed with molecular imaging data involving subcutaneous tumors labeled with genetically expressed iRFP proteins and organ perfusion by optical contrast agents. It is shown that constrained reconstruction is essential for reducing the critical image artifacts associated with inaccurate modeling assumptions. Furthermore, imposing the non-negativity constraint directly on the unmixed distribution of the probe of interest was found to maintain the most robust and accurate reconstruction performance in all experiments.

Performance of a Multispectral Optoacoustic Tomography (MSOT) System equipped with 2D vs. 3D Handheld Probes for Potential Clinical Translation.

A handheld approach to optoacoustic imaging is essential for the clinical translation. The first 2- and 3-dimensional handheld multispectral optoacoustic tomography (MSOT) probes featuring real-time unmixing have recently been developed. Imaging performance of both probes was determined in vitro and in a brain melanoma metastasis mouse model in vivo. T1-weighted MR images were acquired for anatomical reference. The limit of detection of melanoma cells in vitro was significantly lower using the 2D than the 3D probe. The signal decrease was more profound in relation to depth with the 3D versus the 2D probe. Both approaches were capable of imaging the melanoma tumors qualitatively at all time points. Quantitatively, the 2D approach enabled closer anatomical resemblance of the tumor compared to the 3D probe, particularly at depths beyond 3 mm. The 3D probe was shown to be superior for rapid 3D imaging and, thus, holds promise for more superficial target structures.

Multispectral optoacoustic and MRI coregistration for molecular imaging of orthotopic model of human glioblastoma.

Multi-modality imaging methods are of great importance in oncologic studies for acquiring complementary information, enhancing the efficacy in tumor detection and characterization. We hereby demonstrate a hybrid non-invasive in vivo imaging approach of utilizing magnetic resonance imaging (MRI) and Multispectral Optoacoustic Tomography (MSOT) for molecular imaging of glucose uptake in an orthotopic glioblastoma in mouse. The molecular and functional information from MSOT can be overlaid on MRI anatomy via image coregistration to provide insights into probe uptake in the brain, which is verified by ex vivo fluorescence imaging and histological validation. In vivo MSOT and MRI imaging of an orthotopic glioma mouse model injected with IRDye800-2DG. Image coregistration between MSOT and MRI enables multifaceted (anatomical, functional, molecular) information from MSOT to be overlaid on MRI anatomy images to derive tumor physiological parameters such as perfusion, haemoglobin and oxygenation.

Structural and Functional Analysis of Intact Hair Follicles and Pilosebaceous Units by Volumetric Multispectral Optoacoustic Tomography.

Visualizing anatomical and functional features of hair follicle development in their unperturbed environment is key in understanding complex mechanisms of hair pathophysiology and in discovery of novel therapies. Of particular interest is in vivo visualization of the intact pilosebaceous unit, vascularization of the hair bulb, and evaluation of the hair cycle, particularly in humans. Furthermore, noninvasive visualization of the sebaceous glands could offer crucial insight into the pathophysiology of follicle-related diseases and dry or seborrheic skin, in particular by combining in vivo imaging with other phenotyping, genotyping, and microbial analyses. The available imaging techniques are limited in their ability for deep tissue in vivo imaging of hair follicles and lipid-rich sebaceous glands in their entirety without biopsy. We developed a noninvasive, painless, and risk-free volumetric multispectral optoacoustic tomography method for deep tissue three-dimensional visualization of whole hair follicles and surrounding structures with high spatial resolution below 80 µm. Herein we demonstrate on-the-fly assessment of key morphometric parameters of follicles and lipid content as well as functional oxygenation parameters of the associated capillary bed. The ease of handheld operation and versatility of the newly developed approach poise it as an indispensable tool for early diagnosis of disorders of the pilosebaceous unit and surrounding structures, and for monitoring the efficacy of cosmetic and therapeutic interventions.

Metastatic status of sentinel lymph nodes in melanoma determined noninvasively with multispectral optoacoustic imaging.

Sentinel lymph node (SLN) excision is included in various cancer guidelines to identify microscopic metastatic disease. Although effective, SLN excision is an invasive procedure requiring radioactive tracing. Novel imaging approaches assessing SLN metastatic status could improve or replace conventional lymph node excision protocols. In our first-in-human study, we used noninvasive multispectral optoacoustic tomography (MSOT) to image SLNs ex vivo and in vivo in patients with melanoma, to determine metastatic status. MSOT significantly improved the tumor metastasis detection rate in excised SLN (506 SLNs from 214 melanoma patients) compared with the conventional EORTC (European Organisation for Research and Treatment of Cancer) Melanoma Group protocol (22.9% versus 14.2%). MSOT combined with the near-infrared fluorophore indocyanine green reliably visualized SLNs in vivo in 20 patients, up to 5-cm penetration and with 100% concordance with (99m)Tc-marked SLN lymphoscintigraphy. MSOT identified cancer-free SLNs in vivo and ex vivo without a single false negative (189 total lymph nodes), with 100% sensitivity and 48 to 62% specificity. Our findings indicate that a noninvasive, nonradioactive MSOT-based approach can identify and determine SLN status and confidently rule out the presence of metastasis. The study further demonstrates that optoacoustic imaging strategies can improve the identification of SLN metastasis as an alternative to current invasive SLN excision protocols.

Targeting Acidity in Pancreatic Adenocarcinoma: Multispectral Optoacoustic Tomography Detects pH-Low Insertion Peptide Probes In Vivo.

BACKGROUND: pH-low insertion peptides (pHLIP) can serve as a targeting moiety that enables pH-sensitive probes to detect solid tumors. Using these probes in conjunction with multispectral optoacoustic tomography (MSOT) is a promising approach to improve imaging for pancreatic cancer. METHODS: A pH-sensitive pHLIP (V7) was conjugated to 750 NIR fluorescent dye and evaluated as a targeted probe for pancreatic adenocarcinoma. The pH-insensitive K7 pHLIP served as an untargeted control. Probe binding was assessed in vitro at pH 7.4, 6.8, and 6.6 using human pancreatic cell lines S2VP10 and S2013. Using MSOT, semiquantitative probe accumulation was then assessed in vivo with a murine orthotopic pancreatic adenocarcinoma model. RESULTS: In vitro, the V7-750 probe demonstrated significantly higher fluorescence at pH 6.6 compared with pH 7.4 (S2VP10, P = 0.0119; S2013, P = 0.0160), whereas no difference was observed with the K7-750 control (S2VP10, P = 0.8783; S2013, P = 0.921). In the in vivo S2VP10 model, V7-750 probe resulted in 782.5 MSOT a.u. signal compared with 5.3 MSOT a.u. in K7-750 control in tumor (P = 0.0001). Similarly, V7-750 probe signal was 578.3 MSOT a.u. in the S2013 model compared with K7-750 signal at 5.1 MSOT a.u. (P = 0.0005). There was minimal off-target accumulation of the V7-750 probe within the liver or kidney, and probe distribution was confirmed with ex vivo imaging. CONCLUSIONS: Compared with pH-insensitive controls, V7-750 pH-sensitive probe specifically targets pancreatic adenocarcinoma and has minimal off-target accumulation. The noninvasive detection of pH-targeted probes by means of MSOT represents a promising modality to improve the detection and monitoring of pancreatic cancer.

Extending biological imaging to the fifth dimension: evolution of volumetric small animal multispectral optoacoustic tomography.

Despite the ancient discovery of the basic physical phenomenon underlying optoacoustic imaging and tomography [1], the lack of suitable laser sources, ultrasound detection technology, data acquisition, and processing capacities has long hindered the realization of efficient imaging devices. In fact, the first high-quality images from living animals were obtained about a decade ago (Figure 1), which was followed by an exponential growth of technical developments in instrumentation, algorithms, and biomedical applications surrounding this fascinating field. The ability of optoacoustics to probe optical contrast along a wide domain of penetration scales while maintaining excellent spatiotemporal resolution representative of ultrasound imaging, as shown in Figure 2, is unparalleled among the other optical imaging modalities [2], [3].

FMT-PCCT: hybrid fluorescence molecular tomography-x-ray phase-contrast CT imaging of mouse models.

The implementation of hybrid fluorescence molecular tomography (FMT) and X-ray computed tomography (CT) has been shown to be a necessary development, not only for combining anatomical with functional and molecular contrast, but also for generating optical images of high accuracy. FMT affords highly sensitive 3-D imaging of fluorescence bio-distribution, but in stand-alone form it offers images of low resolution. It was shown that FMT accuracy significantly improves by considering anatomical priors from CT. Conversely, CT generally suffers from low soft tissue contrast. Therefore utilization of CT data as prior information in FMT inversion is challenging when different internal organs are not clearly differentiated. Instead, we combined herein FMT with emerging X-ray phase-contrast CT (PCCT). PCCT relies on phase shift differences in tissue to achieve soft tissue contrast superior to conventional CT. We demonstrate for the first time FMT-PCCT imaging of different animal models, where FMT and PCCT scans were performed in vivo and ex vivo, respectively. The results show that FMT-PCCT expands the potential of FMT in imaging lesions with otherwise low or no CT contrast, while retaining the cost benefits of CT and simplicity of hybrid device realizations. The results point to the most accurate FMT performance to date.

Multispectral optoacoustic tomography at 64, 128, and 256 channels.

Optoacoustic (photoacoustic) imaging has already showcased the capacity to offer high-resolution small animal visualization in vivo in a variety of cancer, cardiovascular, or neuroimaging applications. In particular, multispectral optoacoustic tomography (MSOT) has shown imaging along the spectral and the time dimensions, enabling sensing of multiple molecules over time and, more recently, in real time. Furthermore, cross-sectional imaging of at least 20 mm diameter has been showcased in vivo in animals and humans using 64-element curved transducers placed along a single curved line. Herein, we investigated the imaging improvements gained by utilizing a larger number of detectors and inquired whether more detectors will result in measurable image quality improvements. For this reason, we implemented MSOT using 64-, 128-, and 256-element transducers and imaged the same phantoms and animals under similar conditions. Further, corroborated by numerical simulation analysis, our findings quantify the improvements in resolution and overall image quality for the increasing number of detectors used pointing to significant improvements in image quality for the 256 detector array, over 64 or 128 detectors.

Deep-tissue reporter-gene imaging with fluorescence and optoacoustic tomography: a performance overview.

PURPOSE: A primary enabling feature of near-infrared fluorescent proteins (FPs) and fluorescent probes is the ability to visualize deeper in tissues than in the visible. The purpose of this work is to find which is the optimal visualization method that can exploit the advantages of this novel class of FPs in full-scale pre-clinical molecular imaging studies. PROCEDURES: Nude mice were stereotactically implanted with near-infrared FP expressing glioma cells to from brain tumors. The feasibility and performance metrics of FPs were compared between planar epi-illumination and trans-illumination fluorescence imaging, as well as to hybrid Fluorescence Molecular Tomography (FMT) system combined with X-ray CT and Multispectral Optoacoustic (or Photoacoustic) Tomography (MSOT). RESULTS: It is shown that deep-seated glioma brain tumors are possible to visualize both with fluorescence and optoacoustic imaging. Fluorescence imaging is straightforward and has good sensitivity; however, it lacks resolution. FMT-XCT can provide an improved rough resolution of ∼1 mm in deep tissue, while MSOT achieves 0.1 mm resolution in deep tissue and has comparable sensitivity. CONCLUSIONS: We show imaging capacity that can shift the visualization paradigm in biological discovery. The results are relevant not only to reporter gene imaging, but stand as cross-platform comparison for all methods imaging near infrared fluorescent contrast agents.

Multispectral opto-acoustic tomography (MSOT) of the brain and glioblastoma characterization.

Brain research depends strongly on imaging for assessing function and disease in vivo. We examine herein multispectral opto-acoustic tomography (MSOT), a novel technology for high-resolution molecular imaging deep inside tissues. MSOT illuminates tissue with light pulses at multiple wavelengths and detects the acoustic waves generated by the thermoelastic expansion of the environment surrounding absorbing molecules. Using spectral unmixing analysis of the data collected, MSOT can then differentiate the spectral signatures of oxygenated and deoxygenated hemoglobin and of photo-absorbing agents and quantify their concentration. By being able to detect absorbing molecules up to centimeters deep in the tissue it represents an ideal modality for small animal brain imaging, simultaneously providing anatomical, hemodynamic, functional, and molecular information. In this work we examine the capacity of MSOT in cross-sectional brain imaging of mice. We find unprecedented optical imaging performance in cross-sectional visualization of anatomical and physiological parameters of the mouse brain. For example, the potential of MSOT to characterize ischemic brain areas was demonstrated through the use of a carbon dioxide challenge. In addition, indocyanine green (ICG) was injected intravenously, and the kinetics of uptake and clearance in the vasculature of the brain was visualized in real-time. We further found that multiparameter, multispectral imaging of the growth of U87 tumor cells injected into the brain could be visualized through the intact mouse head, for example through visualization of deoxygenated hemoglobin in the growing tumor. We also demonstrate how MSOT offers several compelling features for brain research and allows time-dependent detection and quantification of brain parameters that are not available using other imaging methods without invasive procedures.

Pharmacodynamic characterization of chemopreventive triterpenoids as exceptionally potent inducers of Nrf2-regulated genes.

Synthetic triterpenoids have been developed, which are potent inducers of cytoprotective enzymes and inhibitors of inflammation, greatly improving on the weak activity of naturally occurring triterpenoids. An imidazolide triterpenoid derivative, 1-[2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole (CDDO-Im or TP235), has been previously shown to potently protect against hepatic tumorigenesis, acting in part by inducing cytoprotective genes through Keap1-Nrf2-antioxidant response element (ARE) signaling. In these studies, the pharmacodynamic activity of CDDO-Im is characterized in two distinct lines of ARE reporter mice and by measuring increases in Nqo1 transcript levels as a marker of cytoprotective gene induction. Oral administration of CDDO-Im induces ARE-regulated cytoprotective genes in many tissues in the mouse, including liver, lung, kidney, intestines, brain, heart, thymus, and salivary gland. CDDO-Im induces Nqo1 RNA transcripts in some organs at doses as low as 0.3 mumol/kg body weight (orally). A structure activity evaluation of 15 additional triterpenoids (a) confirmed the importance of Michael acceptor groups on both the A and C rings, (b) showed the requirement for a nitrile group at C-2 of the A ring, and (c) indicated that substituents at C-17 dramatically affected pharmacodynamic action in vivo. In addition to CDDO-Im, other triterpenoids, particularly the methyl ester CDDO-Me (TP155) and the dinitrile TP225, are extremely potent inducers of cytoprotective genes in mouse liver, lung, small intestine mucosa, and cerebral cortex. This pharmacodynamic characterization highlights the chemopreventive promise of several synthetic triterpenoids in multiple target organs.

In vivo modulation of the Parkinsonian phenotype by Nrf2.

Oxidative stress has been implicated in the etiology of Parkinson's disease (PD) and in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) animal model of PD. In this report we show that Nrf2, a transcription factor that regulates the expression of phase 2 and antioxidative enzymes, modulates MPTP neurotoxicity in rodents. Nrf2 knockout and wild-type mice were administered MPTP doses ranging from 20 to 60mg/kg. Seven days after MPTP administration dopamine transporter (DAT) levels were measured using [(125)I]-RTI-121 quantitative autoradiography as an index of dopamine terminal integrity in the striatum. The results indicate that MPTP administration resulted in a greater loss of DAT levels in the striatum of Nrf2 knockout mice than in wild-type at all MPTP doses tested. Activation of the Nrf2 pathway by oral administration of the Nrf2 inducer 3H-1,2-dithiole-3-thione (D3T) to wild-type mice produced partial protection against MPTP-induced neurotoxicity. The protective effect of D3T was not due to a change in MPTP metabolism since the level of the MPTP metabolite MPP+ was not significantly different in the D3T treated striatum relative to vehicle control. Administration of D3T to Nrf2 knockout mice did not protect against MPTP neurotoxicity suggesting that the Nrf2 pathway is necessary for the D3T-mediated attenuation of MPTP neurotoxicity. This study demonstrates the significance of activating intrinsic antioxidative mechanisms in an in vivo model of neurodegeneration. The in vivo activation of the Nrf2 pathway in the brain may be an important strategy to mitigate the effects of oxidative stress in neurodegenerative disorders and neurological disease.