Vogt J, Paul F, Aktas O, Müller-Wielsch K, Dörr J, Dörr S, Bharathi BS, Glumm R, Schmitz C, Steinbusch H, Raine CS, Tsokos M, Nitsch R, Zipp F. Lower motor neuron loss in multiple sclerosis and experimental autoimmune encephalomyelitis. Ann Neurol 2009;66(3):310-22. doi: 10.1002/ana.21719. 

 

Background: Multiple sclerosis (MS) has long been viewed as a demyelinating disease, but increasing evidence points to neuronal degeneration as a major contributor. The distribution and mechanisms of this neuronal loss, especially within the lower motor system, were not well understood. This study examined whether such neuronal loss occurs in MS and its model, experimental autoimmune encephalomyelitis (EAE), and explored underlying immune mechanisms. 

 

Hypothesis: This study hypothesized that lower motor neurons are lost in MS and EAE through an inflammatory mechanism involving T lymphocytes expressing tumor necrosis factor–related apoptosis-inducing ligand (TRAIL). 

 

Methods: The authors conducted electrophysiological assessments in 69 MS patients and 75 controls and quantified neurons in human spinal cord, cortex and mouse EAE tissue using high-precision design-based stereology with Stereo Investigator. Neuronal subtypes and immune cells were identified by morphology and immunostaining. 

 

Results: Electrophysiological data revealed reduced muscle action potentials and motor unit numbers in MS patients. Stereological analysis showed a pronounced neuronal loss – approximately 75% in the spinal cord, including 48% α-motor neuron, 81% γ-motor neuron and 67% interneuron reduction. In EAE, similar neuron loss occurred during the relapse phase and was prevented when TRAIL-deficient T cells were used, indicating TRAIL-mediated immune neurodegeneration. 

 

Conclusions: The study demonstrated that substantial lower motor neuron loss occurs in MS and EAE, mediated by TRAIL-expressing T cells. These findings identify inflammatory neurodegeneration of spinal motor neurons as a key contributor to MS pathology. 

Couto B, Forrest SL, Fearon C, Lee S, Knott S, Li J, Fox SH, Tartaglia MC, Lang AE, Kovacs GG. Midbrain cytotoxic T cells as a distinct neuropathological feature of progressive supranuclear palsy. Brain 2025;148(8):2650-2657. doi: 10.1093/brain/awaf135.

 

Background: Progressive supranuclear palsy (PSP) is a neurodegenerative tauopathy defined by four-repeat tau deposition in neurons and glia, particularly affecting the substantia nigra and midbrain tegmentum. Unlike infectious or autoimmune encephalitides, neurodegenerative diseases generally lack lymphocytic infiltrates. However, immune activation and altered T-cell profiles have been reported in PSP, suggesting a potential autoimmune contribution.

 

Hypothesis: This study hypothesized that cytotoxic T-cell infiltration is a distinctive neuropathological feature of PSP, potentially linked to tau pathology and microglial activation, and greater in PSP than in Parkinson’s disease (PD) or control brains.

 

Methods: The authors analyzed post mortem brain samples from nine PSP patients, ten PD patients and six controls. Serial midbrain sections were immunostained for phosphorylated tau, α-synuclein, CD8 and HLA-DR. Cytotoxic T cells were quantified stereologically using Stereo Investigator with an unbiased Fractionator probe, and microglial and protein-pathology loads were measured via HALO image analysis. Statistical comparisons and correlations assessed relationships among CD8 counts, microglia, and pathology load.

 

Results: CD8-positive cell counts in the substantia nigra were significantly higher in PSP than PD or controls. CD8 cells often contacted neurons in PSP, and their density was greatest in the red nucleus/superior cerebellar peduncle. Microglial activation was also higher in PSP than in controls. CD8-cell counts were independent of disease duration, age or tau load.

 

Conclusions: These findings identify midbrain cytotoxic T-cell infiltration as a distinctive feature of PSP, suggesting an autoimmune component in its pathogenesis and providing a potential target for future therapeutic exploration.

Storkebaum E, Lambrechts D, Dewerchin M, Moreno-Murciano MP, Appelmans S, Oh H, Van Damme P, Rutten B, Man WY, De Mol M, Wyns S, Manka D, Vermeulen K, Van Den Bosch L, Mertens N, Schmitz C, Robberecht W, Conway EM, Collen D, Moons L, Carmeliet P. Treatment of motoneuron degeneration by intracerebroventricular delivery of VEGF in a rat model of ALS. Nat Neurosci 2005;8(1):85-92. doi: 10.1038/nn1360.

 

Background: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by progressive loss of spinal and bulbar motoneurons. Previous attempts using neurotrophic factors failed to yield significant therapeutic benefit, partly due to limited delivery to target neurons. Vascular endothelial growth factor (VEGF) had shown neuroprotective properties in genetic and viral delivery models.

 

Hypothesis: This study hypothesized that continuous intracerebroventricular (i.c.v.) delivery of recombinant VEGF protects motoneurons and prolong survival in rat models of ALS.

 

Methods: The authors stereotactically implanted osmotic minipumps to infuse VEGF into the lateral ventricle of SOD1^G93A rats and used radiolabeled VEGF to assess biodistribution. Motor behavior and survival were monitored. For histological analysis, motoneurons in the facial nucleus and spinal cord were quantified using design-based stereology with Stereo Investigator, and axons were assessed on toluidine blue–stained ventral root sections.

 

Results: Continuous i.c.v. VEGF delivery delayed disease onset, improved motor performance and extended survival by up to 22 days. Stereological analysis revealed that VEGF doubled facial motoneuron numbers and preserved large cervical α-motoneurons and ventral root axons. VEGF also maintained neuromuscular junction innervation.

 

Conclusions: Continuous i.c.v. VEGF administration confers robust neuroprotection in ALS models, preserving motoneurons and neuromuscular connectivity and demonstrating therapeutic potential for human neurodegenerative disease.

Wu J, Kharebava G, Piao C, Stoica BA, Dinizo M, Sabirzhanov B, Hanscom M, Guanciale K, Faden AI. Inhibition of E2F1/CDK1 pathway attenuates neuronal apoptosis in vitro and confers neuroprotection after spinal cord injury in vivo. PLoS One 2012;7(7):e42129. doi: 10.1371/journal.pone.0042129.

 

Background: Neuronal apoptosis contributes significantly to secondary tissue damage after spinal cord injury (SCI). The E2F1/CDK1 signaling pathway, known to regulate both cell proliferation and apoptosis, is implicated in neuronal death in various neurodegenerative models, but its specific role after SCI has not been established. Understanding how this pathway influences neuronal apoptosis could identify potential therapeutic targets for SCI.

 

Hypothesis: This study hypothesized that activation of the E2F1/CDK1 signaling pathway contributes to neuronal apoptosis after spinal cord injury and that inhibition of this pathway would attenuate neuronal death and improve neuroprotection.

 

Methods: The authors used both in vitro neuronal cultures and an in vivo rat contusion SCI model. Protein expression and apoptosis were assessed by Western blotting, immunohistochemistry and TUNEL staining. Neuronal survival was quantified using unbiased stereological cell counts analyzed with Stereo Investigator. E2F1 or CDK1 expression was manipulated with shRNA, and apoptosis was pharmacologically modulated with CDK inhibitors such as roscovitine and CR8.

 

Results: SCI rapidly upregulated E2F1 and CDK1 protein expression, which remained elevated for several days. Their downstream targets, Bim and c-Myb, as well as biochemical markers of apoptosis, were increased. Inhibition of E2F1/CDK1 by shRNA or CR8 reduced apoptotic markers, decreased TUNEL-positive neurons and significantly increased surviving neurons five weeks after injury.

 

Conclusions: Activation of the E2F1/CDK1 pathway promotes neuronal apoptosis after spinal cord injury, while its inhibition confers significant neuroprotection. Targeting this signaling cascade may represent an effective therapeutic strategy for SCI.

Zhang QW, Deng XX, Sun X, Xu JX, Sun FY. Exercise promotes axon regeneration of newborn striatonigral and corticonigral projection neurons in rats after ischemic stroke. PLoS One 2013;8(11):e80139. doi: 10.1371/journal.pone.0080139.

 

Background: Ischemic stroke often causes degeneration of striatal and cortical neurons and associated motor deficits. Previous studies showed that new neurons generated after stroke can form projections to the substantia nigra, and that physical exercise promotes neurogenesis and functional recovery. However, whether exercise also enhances axon regeneration of newborn projection neurons in the ischemic adult brain remained unclear.

 

Hypothesis: This study hypothesized that post-stroke treadmill exercise promotes axon regeneration of newborn striatonigral and corticonigral projection neurons, thereby improving motor function recovery after ischemic injury.

 

Methods: The authors subjected adult male rats to transient middle cerebral artery occlusion and subsequently provided treadmill exercise for 30 minutes daily from day 5 to day 28 post-stroke. Fluorogold was injected into the substantia nigra to trace projection neurons, and a GFP-retroviral vector was used to label newborn neurons. Immunohistochemistry, confocal microscopy and unbiased stereological cell counting assisted with Stereo Investigator were performed to quantify labeled neurons. Motor recovery was assessed with neurological scoring and rotarod performance.

 

Results: Exercise significantly improved motor function and increased the numbers of NeuN+, FG+, and GFP+-FG+ neurons in the striatum and cortex ipsilateral to ischemia. It also enhanced tyrosine hydroxylase-positive dopaminergic neurons in the substantia nigra, elevated BDNF, VEGF and synapsin expression, and reduced Nogo-A levels.

 

Conclusions: Post-stroke treadmill exercise promotes axon regeneration of newborn projection neurons and dopaminergic cell survival, enhancing neural repair and motor recovery in ischemic rat brains.

Henny P, Brown MT, Micklem BR, Magill PJ, Bolam JP. Stereological and ultrastructural quantification of the afferent synaptome of individual neurons. Brain Struct Funct 2014;219(2):631-640. doi: 10.1007/s00429-013-0523-9.

 

Background: Understanding how neurons integrate information depends on knowing the number and distribution of their synaptic inputs. While classical neuroanatomy revealed neuronal morphology, unbiased quantitative data on synaptic organization remain limited. This study in the rat brain combined light and electron microscopy with stereological sampling to obtain precise, three-dimensional measurements of synaptic inputs onto single neurons, providing a foundation for linking structure and function.

 

Hypothesis: This study hypothesized that combining juxtacellular labeling, digital neuronal reconstruction and stereological sampling at the ultrastructural level allows unbiased and accurate estimation of the total number and somato-dendritic distribution of synaptic inputs received by individual neurons.

 

Methods: The authors labeled single neurons in vivo in rats, sectioned the brain at constant thickness and performed random systematic sampling. Neuronal morphology was digitally reconstructed using Neurolucida, and serial ultrathin sections were analyzed with electron microscopy. Synapses were identified and counted directly from the electron microscopic images using the Stereo Investigator Optical Fractionator probe. This integrated stereological and reconstruction approach enabled estimation of total synapse number and mapping of their distribution along the soma and dendrites.

 

Results: A representative neuron was estimated to receive 10,488 synapses with a coefficient of error of 0.09. Reduced sampling decreased precision. Synapses were most numerous on proximal, low-order dendrites but densest on the soma and distal branches. Estimates were consistent with previously reported stereological values in other brain regions.

 

Conclusions: This protocol provides a reliable and unbiased framework for quantifying and mapping the afferent synaptome of single neurons, enabling structure–function correlations at the cellular level and supporting realistic computational modeling of neuronal processing.

Riudavets MA, Iacono D, Resnick SM, O’Brien R, Zonderman AB, Martin LJ, Rudow G, Pletnikova O, Troncoso JC. Resistance to Alzheimer’s pathology is associated with nuclear hypertrophy in neurons. Neurobiol Aging 2007;28(10):1484-1492. doi: 10.1016/j.neurobiolaging.2007.05.005.

 

Background: Alzheimer’s disease (AD) pathology is sometimes present in individuals who remain cognitively normal, suggesting that certain neuronal characteristics may confer resilience to the disease. Understanding the structural features of neurons that allow resistance to AD-related lesions could help explain why some brains remain cognitively intact despite heavy amyloid and tau pathology.

 

Hypothesis: This study hypothesized that neurons in cognitively normal individuals with AD pathology (asymptomatic AD) exhibit specific morphometric changes that may reflect adaptive or protective mechanisms against neurodegeneration.

 

Methods: The authors examined post mortem brains from control, asymptomatic AD, mild cognitive impairment (MCI) and AD-dementia subjects. They analyzed neuronal and nuclear volumes in the anterior cingulate gyrus and hippocampal CA1 using stereological techniques with Stereo Investigator and the Vertical Nucleator and Area-Fraction-Fractionator probes. Measurements were performed on cresyl violet–stained sections, with additional quantification of amyloid-β and phosphorylated tau immunoreactivity.

 

Results: Compared to controls, asymptomatic AD brains showed no neuronal atrophy in either region. In contrast, neuronal cell body volumes were significantly reduced in MCI and AD. Asymptomatic AD neurons displayed marked nuclear hypertrophy, with significantly larger nuclear volumes than controls, MCI and AD, while cerebellar Purkinje cells showed no group differences. Amyloid and tau burdens were comparable between asymptomatic AD and MCI, but both were higher in AD.

 

Conclusions: Nuclear hypertrophy in asymptomatic AD neurons may represent an early adaptive response to AD pathology, reflecting enhanced metabolic or compensatory activity that delays neuronal degeneration and preserves cognitive function.

Liu P, Reichl JH, Rao ER, McNellis BM, Huang ES, Hemmy LS, Forster CL, Kuskowski MA, Borchelt DR, Vassar R, Ashe KH, Zahs KR. Quantitative comparison of dense-core amyloid plaque accumulation in amyloid-β protein precursor transgenic mice. J Alzheimers Dis 2017;56(2):743-761. doi: 10.3233/JAD-161027.

 

Background: Alzheimer’s disease involves the accumulation of amyloid-β (Aβ) plaques, but transgenic mouse models expressing human Aβ precursor protein (AβPP) vary widely in plaque development and distribution. Comparing these models quantitatively can clarify how plaque pathology progresses and how well each model replicates human disease.

 

Hypothesis: This study hypothesized that distinct AβPP transgenic mouse lines would show significant differences in the timing, magnitude and regional distribution of dense-core amyloid plaque accumulation.

 

Methods: The authors examined four AβPP transgenic lines—5XFAD, APPSwePS1ΔE9, Tg2576 and rTg9191—using Thioflavin S staining to visualize dense-core plaques in cortex and hippocampus. Plaque burdens and areas were quantified stereologically with the Stereo Investigator system’s Area Fractionator and Nucleator modules. Additional imaging was performed with an Axio Imager microscope, an AxioCam digital camera and a Nikon TiE deconvolution microscope.

 

Results: Dense-core plaques appeared earliest and most abundantly in 5XFAD mice, followed by APPSwePS1ΔE9, Tg2576 and rTg9191. At 15 months, cortical plaque burden in 5XFAD mice was about 4.5 times higher than in 21-month-old Tg2576 and 15 times higher than in 24-month-old rTg9191. Plaque-size distributions changed with age and brain region. In three mouse lines, cortical plaque burdens eventually exceeded those measured in human Alzheimer’s cortex.

 

Conclusions: The study concludes that dense-core plaque accumulation differs by more than an order of magnitude among AβPP transgenic lines, reflecting genetic and biochemical variability. These quantitative differences highlight the need for careful model selection and standardized measurement methods when interpreting Aβ-related pathology in AD research.

Kreczmanski P, Heinsen H, Mantua V, Woltersdorf F, Masson T, Ulfig N, Schmidt-Kastner R, Korr H, Steinbusch HW, Hof PR, Schmitz C. Microvessel length density, total length, and length per neuron in five subcortical regions in schizophrenia. Acta Neuropathol 2009;117(4):409-421. doi: 10.1007/s00401-009-0482-7.

 

Background: Schizophrenia has been associated with metabolic and vascular abnormalities, but direct structural evidence for microvascular alterations in the brain remains limited. Because adequate blood supply is essential for neuronal function, subtle vascular changes could contribute to the pathophysiology of the disorder.

 

Hypothesis: This study hypothesized that patients with schizophrenia show alterations in microvessel length density, total microvessel length or microvessel length per neuron in subcortical brain regions compared to healthy controls.

 

Methods: The authors examined post mortem brains from 13 male patients with schizophrenia and 13 age-matched male controls. Using design-based stereology with the Spaceballs probe implemented in Stereo Investigator and analyzed through a motorized microscope system equipped with an electronic microcator and CCD camera, they estimated microvessel length density in the caudate nucleus, putamen, nucleus accumbens, mediodorsal thalamic nucleus and lateral amygdala.

 

Results: No significant differences were found between schizophrenia and control groups in microvessel length density, total microvessel length or microvessel length per neuron in any of the investigated regions. Microvessel length parameters were also unrelated to illness duration or neuronal density.

 

Conclusions: The findings indicate that microvascular structure in key subcortical regions is preserved in schizophrenia, suggesting that functional vascular or metabolic abnormalities arise independently of measurable structural microvessel changes.

Gama Sosa MA, Gasperi RD, Rocher AB, Wang AC, Janssen WG, Flores T, Perez GM, Schmeidler J, Dickstein DL, Hof PR, Elder GA. Age-related vascular pathology in transgenic mice expressing presenilin 1-associated familial Alzheimer’s disease mutations. Am J Pathol 2010;176(1):353-368. doi: 10.2353/ajpath.2010.090482.

 

Background: Familial Alzheimer’s disease (FAD) is associated with presenilin 1 (PS1) mutations that may affect neuronal and vascular integrity. Vascular pathology, including basement membrane alterations and capillary degeneration, is a frequent but poorly understood feature of Alzheimer’s disease. This study examined whether PS1 mutations alone are sufficient to induce vascular abnormalities in the absence of amyloid plaque deposition.

 

Hypothesis: This study hypothesized that expression of PS1 FAD mutations in neurons disrupts vascular structure and integrity, producing age-related microvascular pathology similar to that seen in Alzheimer’s disease.

 

Methods: The authors used transgenic mice expressing either wild-type or FAD-mutant human PS1 (M146V and P117L). Brain vasculature was analyzed in 50-µm coronal sections immunostained for collagen IV. Stereologic quantification of hippocampal vasculature was performed using a Zeiss Axioplan 2 microscope and Stereo Investigator with the Space Balls probe. Electron microscopy was used to assess ultrastructural changes.

 

Results: PS1 FAD-mutant mice exhibited age-dependent vascular attrition, decreased hippocampal capillary length density and thickened, dystrophic basement membranes. Electron microscopy revealed degenerated endothelial cells, irregular lumens and string vessel formation, despite intact surrounding neuropil. No congophilic amyloid was detected.

 

Conclusions: Neuronal expression of PS1 FAD mutations alone induces microvascular pathology, suggesting that mutant PS1 disrupts neuron–vascular signaling and contributes to Alzheimer’s disease–related vascular degeneration independent of amyloid deposition.