Acute and non-resolving inflammation associate with oxidative injury after human spinal cord injury

Abstract

Traumatic spinal cord injury is a devastating insult followed by progressive cord atrophy and neurodegeneration. Dysregulated or non-resolving inflammatory processes can disturb neuronal homeostasis and drive neurodegeneration. Here, we provide an in-depth characterization of innate and adaptive inflammatory responses as well as oxidative tissue injury in human traumatic spinal cord injury lesions compared to non-traumatic control cords. In the lesion core, microglia were rapidly lost while intermediate (co-expressing pro- as well as anti-inflammatory molecules) blood-borne macrophages dominated. In contrast, in the surrounding rim, TMEM119+ microglia numbers were maintained through local proliferation and demonstrated a predominantly pro-inflammatory phenotype. Lymphocyte numbers were low and mainly consisted of CD8+ T cells. Only in a subpopulation of patients, CD138+/IgG+ plasma cells were detected, which could serve as candidate cellular sources for a developing humoral immunity. Oxidative neuronal cell body and axonal injury was visualized by intracellular accumulation of amyloid precursor protein (APP) and oxidized phospholipids (e06) and occurred early within the lesion core and declined over time. In contrast, within the surrounding rim, pronounced APP+/e06+ axon-dendritic injury of neurons was detected, which remained significantly elevated up to months/years, thus providing mechanistic evidence for ongoing neuronal damage long after initial trauma. Dynamic and sustained neurotoxicity after human spinal cord injury might be a substantial contributor to (i) an impaired response to rehabilitation; (ii) overall failure of recovery; or (iii) late loss of recovered function (neuro-worsening/degeneration).

Keywords: adaptive immunity; blood-derived macrophages; microglia; oxidative injury; spinal cord injury.

Figure 1

Spatiotemporal characterization of neuropathological features in human SCI lesions. The sequence of neuropathological alterations was grouped into four stages (I–IV). An overview of the progressive lesion evolution is illustrated by haematoxylin and eosin (H&E)-stained spinal cord cross-sections (middle row) accompanied by higher-resolution details of the corresponding lesion core (left) and surrounding rim (right). Anterior spinal cord is facing downwards, while dorsal cord is directed upwards. Stage I (1–3 days post-SCI): (A) Distortion and disruption of parenchyma and inherent vessels with ensuing haemorrhage and leukodiapedesis (extravasation, arrows) in the lesion core. Hyperpolarized red blood cell are detected outside of the neurovascular unit in a radial manner in close vicinity to the damaged vessels (B). Abundant swollen hypertrophic β-APP⁺ axonal spheroids (black arrow) indicate axonal injury. These ballooned retraction bulbs indicate severe cytoskeletal disruption and are intermingled with β-APP⁺ axons displaying features (dotted segregated β-APP⁺ alignments; green arrow) in line with axonal fragmentation (Williams et al., 2014) (C). (D) In the lesion-surrounding white matter, there is evidence of beginning myelin damage (demyelination) with early CNPase loss (black dot), while (E) PLP still remains preserved. Stage II (4–21 days post-SCI): Tissue necrosis with massive, macrophage infiltration into the lesion core. Emerging spongiotic tissue changes (arrows) reflect actively ongoing debris removal (F) evidenced by CD68⁺ macrophages containing large lysosomal vacuoles (arrows) (G). Non-phagocytosed extravasated erythrocytes are, however, still present (F, green arrow). (H) A demarcation between lesion core (asterisk) and lesion rim is visible since areas of impaired axoplasmic transport in the rim show massive accumulation of β-APP⁺ axonal spheroids. Hypertrophic retraction bulbs (arrow) are detectable in close juxtaposition to the lesion. (I) Density of CD68⁺ monocytes/macrophages is lower and the lysosomes smaller. Stage III (22–90 days post-SCI): (J and K) Progressive, enlarged spongiotic changes (asterisks) emerging towards the beginning demarcation of a cystic cavitation In the surrounding rim, remaining axonal β-APP+ spheroids (L) are still present; however, in reduced numbers compared to the preceding stage II. Signs of axonal fragmentation are no longer detected. (M) β-APP⁺ axons are surrounded by reactive gliosis composed of hypertrophic activated GFAP⁺ astrocytes reflective of maturated lesion organization (arrows). Hypertrophic GFAP⁺ astrocytes extend vascular end-feet with pronounced bouton-type appearance (green arrows) to support vascular integrity (stabilization of the neurovascular unit) (M). Stage IV (22–90 days post-SCI): (N) Cystic cavitation (syrinx) and surrounding scar formation is composed of an hypercellular rim, demonstrating declining cell numbers (gradient, black dot), further extending towards the core (asterisk) into an area of extracellular matrix deposition characterized by hypocellularity (black dot). (O) The scar contains non-resolving, activated CD68⁺ macrophages, which form an inflammatory ‘layer’ confined to the immediate border between the hypercellular and hypocellular regions with declining cell numbers towards the syrinx core. Non-resolving lipid-laden (foamy) CD68⁺ macrophages form compartmentalized clusters ‘locked’ into the immediate scar (arrow). CD68⁺ monocytes/macrophages also form clusters in Virchow-Robin-alike spaces (green arrow) pointing to a chronic immunological activation or drainage (Wardlaw et al., 2020). (P and Q) The rim illustrates massive reactive GFAP⁺ astrogliosis with some remaining evenly distributed CD68⁺ macrophages. Of note, also the rim represents an area of higher immune alertness characterized by higher numbers of CD68⁺ myeloid cells even until chronic stages. Spinal cord tissue from controls did not contain any pathological changes (not shown). Scale bars = 25 µm in A–C, F, G, I–M, P and Q; 250 µm in D and E; and 125 µm in H, N and O.

Figure 2

Quantitative analysis of cellular markers in the spinal cord lesion core. Quantitation of PMNs and various microglia/macrophage marker expression in the core of SCI lesions at different lesion stages (I–IV) as well as in non-traumatic control tissue. Data represent actual numbers of cells/mm². (A) PMNs counted in haematoxylin and eosin staining, (B) pan-microglia/macrophage marker Iba-1, (C) the microglia-specific marker TMEM119, (D) the ‘homeostatic’ microglia marker P2RY12; and the microglia activation markers (E) p22phox, (F) CD68, (G) HLA-DR, (H) CD86, (I) iNOS, (J) CD163, (K) CD206 and DAB-enhanced TBB staining for the detection of non-haem iron (L). *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.

Figure 3

Quantitative analysis of cellular markers in the spinal cord lesion rim. Quantitation of PMNs and various microglia/macrophage marker expression in the core of SCI lesions at different lesion stages (I–IV) as well as in non-traumatic control tissue. Data represent actual numbers of cells/mm². (A) PMNs counted in haematoxylin and eosin staining, (B) pan-microglia/macrophage marker Iba-1, (C) the microglia-specific marker TMEM119, (D) the ‘homeostatic’ microglia marker P2RY12; and the microglia activation markers (E) p22phox, (F) CD68, (G) HLA-DR, (H) CD86, (I) iNOS, (J) CD163, (K) CD206 and DAB-enhanced Turnbull Blue (TBB) staining for the detection of non-haem iron (L). *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.

Figure 4

Loss of ‘homeostatic’ microglia within the lesion core and rim. Segregation of infiltrating monocytes towards the core while microglia persist within the rim. Distinct and facetted microglia and macrophage response to human traumatic SCI (A). Almost all microglia are double positive for Iba-1 (green) and TMEM119 (red) as well as (B) co-express P2RY12 (green) and TMEM119 (red) in the spinal cord of non-traumatic controls. (C) The rim of the lesions contains high numbers of Iba-1 (green) TMEM119 (red) double-positive cells, while (D) TMEM119⁺ (red) cells are massively reduced in the lesion core. Likewise, (E) sparse TMEM119 (red) P2RY12 (green) double-positive cells are still detectable in the rim, while (F) they are completely lost in the lesion core. In the lesion rim, the majority of microglia (TMEM119⁺ cells; red), expresses pro-inflammatory markers e.g. (G) p22phox (blue) (stage II) or (I) the phagocytosis-associated marker CD68 (blue); within the lesion core, hardly any myeloid cells expressing pro-inflammatory markers, e.g. (H) p22phox, co-express the microglia marker TMEM119. (J) Many microglia (TMEM119⁺, red) within the rim display nuclear co-expression of the proliferation marker proliferating cell nuclear antigen (PCNA; blue). (K) An example of an SCI lesion; within the core (left side), numerous pro-inflammatory (p22phox; blue) and anti-inflammatory (CD206; red) double-positive macrophages are present, whereas pro-inflammatory activation predominates within the rim (right side). (L and M) Higher magnification of lesion (L) core and (M) rim presented in K. (N) Same lesion presented in K showing sparse TMEM119 and CD206 double-positive cells within the lesion core (left side), whereas in the rim (right cells, CD206⁺ cells are hardly present and TMEM119 single-positive cells prevail. (O and P) Higher magnification of lesion (O) core and (P) rim presented in N. (Q) In the core of stage II lesions, numerous CD163⁺ macrophages are present; similarly, CD163⁺ microglia are observed in the rim (R). Low numbers of TBB⁺ cells were found in the core (S) as well as in the lesion rim (T). Scale bars = 25 µm. Ctl = Control; C = Core; I–IV = stages; R = Rim.

Figure 5

Cytokine expression in traumatic SCI. Numerous IL-18⁺ microglia and macrophages in the rim (A) and core (B) of SCI lesions. In contrast, TGF-β expression is restricted to astrocytes (C and D) in the lesion rim. Fluorescent triple stainings confirm TGF-β expression is restricted to GFAP⁺ astrocytes (E). Conformational triple stainings demonstrating IL-18 expression in TMEM119⁺ microglia in the rim (F) and in TMEM119⁻ macrophages in the core (G). While in the lesion rim pro-inflammatory IL-18⁺ microglia cells prevail (H), anti-inflammatory polarized CD206⁺ macrophages (I) are devoid of IL-18. All pictures are taken from stage II patients except C and D, which were taken from stage III. Scale bar = 25 µm.

Figure 6

Moderate T cell and sparse B cell infiltration into the spinal cord after human traumatic SCI. Profile of total lymphocytes numbers populating the injured SCI over time. (A–E) Quantitation of the expression of the T cell markers CD3 (A) and CD8 (B), the B cell markers CD20 (C) and CD79a (D) and the plasma cell marker CD138 (E) in SCI lesions at different lesion stages (I–IV) compared with non-traumatic control tissue. Data represent numbers of cells/mm² *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. (F) T cells were scarce, mostly perivascular, when observed in control patients. (G–I) Infiltrating CD3⁺ cells (G) in SCI are mainly MHC class I-restricted CD8⁺ cells (H), whereas only scattered CD4⁺ cells (I) were present in the injured neuropil. (J and K) Compared to T cells, single numbers of B cells mostly confined to perivascular areas were observed. (L–N) CD138⁺ plasma cells in a case of SCI was observed, which were identified as IgG positive plasma cells though double-labelling (N). (O) C9neo complement deposition of the same SCI case as in L–N. Scale bar = 25 µm.

Figure 7

Oxidative injury after SCI affects spinal neurons. Presence of oxidized phospholipids (e06 immunoreactivity) in neurons has been associated with beading and fragmentation of cell processes in the human CNS (Fischer et al., 2012) and occurs after SCI. (A and B) Percentage of positive area covered by e06 immunoreactivity within the lesion core (A) and lesion rim (B). (C) Immunoreactivity of e06 in lipofuscin granules in non-traumatic control spinal cords. (D) Few e06⁺ axonal bulbs within the lesion core during the first 3 days post-SCI (stage I). (E) Several e06⁺ axonal bulbs within the rim of chronic spinal cord lesions (stage IV). (F) At stage II, many APP⁺ axonal bulbs are present in the lesion rim, where oxidized phospholipids also accumulate (G). (H) Co-accumulation of oxidized phospholipids and APP in ballooned axonal spheroids and retraction bulbs. (I) Macrophages with granular cytoplasmic reactivity for oxidized phospholipids in the SCI core. (J) Intense e06 immunoreactivity in a neuron showing signs of degeneration and beading/fragmentation of cell processes. (K) Massive accumulation of oxidized phospholipids (e06⁺) in a degenerating neuron surrounded from and in contact with pro-inflammatory p22phox⁺ microglia (blue). (L) e06⁺ neuron undergoing central chromatolysis in contact with activated pro-inflammatory p22phox⁺ microglia. Scale bar = 25 µm.